Compositions and methods for regulating bacterial pathogenesis

ABSTRACT

This invention provides a purified extracellular bacterial autoinducer-2 signaling molecule, the production of which is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. The signaling molecule stimulates LuxQ luminescence genes, and is believed also to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. This invention also provides is a new class of bacterial genes involved in the biosynthesis of the signaling molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/110,570, filed Dec. 2, 1998, the disclosure ofwhich is incorporated herein by reference.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

[0002] Pursuant to 35 U.S.C. §202(c), it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Science Foundation, GrantNo. MCB-9506033.

FIELD OF THE INVENTION

[0003] This invention relates to the field of bacterial diseases ofhumans and other mammals. In particular, the invention provides novelgenes and signaling factors involved in inducing pathogenesis in certainbacteria, and methods for controlling such pathogenesis throughmanipulation of those factors and genes.

BACKGROUND OF THE INVENTION

[0004] Several publications are referenced in this application to morefully describe the state of the art to which this invention pertains.The disclosure of each such publication is incorporated by referenceherein.

[0005] The control of gene expression in response to cell density, orquorum sensing, was first described in the marine luminous bacteriaVibrio fischeri and Vibrio harveyi. This phenomenon has recently becomerecognized as a general mechanism for gene regulation in many Gramnegative bacteria. Quorum sensing bacteria synthesize, release, andrespond to specific acyl-homoserine lactone signaling molecules calledautoinducers to control gene expression as a function of cell density.In all acyl-homoserine lactone quorum sensing systems described to date,except that of V. harveyi, the autoinducer synthase is encoded by a genehomologous to luxI of V. fischeri, and response to the autoinducer ismediated by a transcriptional activator protein encoded by a genehomologous to luxR of V. fischeri (Bassler and Silverman, in Twocomponent Signal Transduction, Hoch et al., eds, Am. Soc. Microbiol.Washington D.C., pp 431-435, 1995). In contrast, V. harveyi has twoindependent density sensing systems (called Signaling Systems 1 and 2),and each is composed of a sensor-autoinducer pair. V. harveyi SignalingSystem 1 is composed of Sensor 1 and autoinducer 1 (AI-1), and thisautoinducer is N-(3-hydroxybutanoyl)-L-homoserine lactone (see Bassleret al., Mol. Microbiol. 9: 773-786, 1993). V. harveyi Signaling System 2is composed of Sensor 2 and autoinducer 2 (AI-2) (Bassler et al., Mol.Microbiol. 13: 273-286, 1994). The structure of AI-2 heretofore has notbeen determined, nor have the gene(s) involved in biosynthesis of AI-2been identified. Signaling System 1 is a highly specific system proposedto be used for intra-species communication and Signaling System 2appears to be less species-selective, and is hypothesized to be forinter-species communication (Bassler et al., J. Bacteriol. 179:4043-4045, 1997).

[0006] Reporter strains of V. harveyi have been constructed that arecapable of producing light exclusively in response to AI-1 or to AI-2(Bassler et al., 1993, supra; Bassler et al., 1994, supra). V. harveyireporter strains have been used to demonstrate that a few species ofbacteria produce stimulatory substances that mimic the action of AI-2(Bassler et al., 1997, supra).

[0007] Quorum sensing in V. harveyi, mediated by Signaling Systems 1 and2, triggers the organisms to bioluminesce at a certain cell density.These same signaling systems, particularly Signaling System 2, arebelieved to trigger other physiological changes in V. harveyi and otherbacteria possessing the same signaling system. Thus, it would be anadvance in the art to identify and characterize the signaling factorautoinducer-2 and the genes encoding the proteins required for itsproduction. Such an advance would provide a means to identify a novelclass of compounds useful for controlling mammalian enteric orpathogenic bacteria.

SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, it has now beendiscovered that a variety of bacterial species, some of them mammalianpathogens, secrete an organic signaling molecule that stimulates theexpression of luminescence in the V. harveyi Signaling System 2bioassay. The molecule secreted by these organisms mimics V. harveyiAI-2 in its physical and functional features. The production in bacteriaof this novel signaling molecule is regulated by changes inenvironmental conditions associated with a shift from a free-livingexistence to a colonizing or pathogenic existence in a host organism.Thus, in addition to stimulating luminescence genes (specificallyluxCDABE) in V. harveyi, the signaling molecule is expected to stimulatea variety of pathogenesis related genes in the bacterial species thatproduce it. A highly purified form of the signaling molecule is providedin the present invention. Also provided is a new class of bacterialgenes involved in the biosynthesis of the signaling molecule.

[0009] According to one aspect, the present invention provides anisolated bacterial extracellular signaling factor comprising at leastone molecule that is polar and uncharged, and having an approximatemolecular weight of less than 1,000 kDa, wherein said factor interactswith LuxQ protein thereby inducing expression of a Vibrio harveyi operoncomprising luminescence genes luxCDABE. In a preferred embodiment, thefactor possesses a specific activity wherein about 0.1 to 1.0 mg of apreparation of the factor stimulates about a 1,000-fold increase inluminescense, as measured in a bioassay using a V. harveyi Sensor 2+reporter strain. In a particularly preferred embodiment, the factor ispurified in such a way that it possesses a specific activity whereinabout 1 to 10 μg of a preparation of the factor stimulates about a1,000-fold increase in luminescence, as measured in a bioassay using aV. harveyi Sensor 2+ reporter strain.

[0010] The signaling factor of the invention is produced by a variety ofbacteria, including but not limited to: Vibrio harveyi, Vibrio cholerae,Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum,Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium,Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borreliaburgfdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersiniapestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacteriumtuberculosis, Enterococcus faecalis, Streptococcus pneumoniae,Streptococcus pyogenes and Staphylococcus aureus.

[0011] In another aspect, the invention provides an isolated bacterialsignaling factor having the formula:

[0012] In another aspect, the invention provides a method foridentifying a compound that regulates the activity of a signaling factorby contacting the signaling factor with the compound, measuring theactivity of the signaling factor in the presence of the compound andcomparing the activity of the signaling factor obtained in the presenceof the compound to the activity of the signaling factor obtained in theabsence of the compound and identifying a compound that regulates theactivity of the signaling factor.

[0013] In yet another aspect, the invention provides a method fordetecting an autoinducer molecule in a sample by contacting the samplewith a bacterial cell, or extract thereof, comprising biosyntheticpathways that produce a detectable amount of light in response to anexogenous autoinducer, the bacterial cell having at least two distinctalterations in gene loci that participate in autoinducer pathways,wherein a first alteration in a gene locus comprises an alteration thatinhibits detection of a first autoinducer and wherein a secondalteration in a gene locus comprises an alteration that inhibitsproduction of a second autoinducer and measuring light produced by thebacterial cell, or extract thereof.

[0014] In another aspect, the invention provides a bacterial cell havingat least two distinct alterations in gene loci that participate inautoinducer pathways, wherein a first alteration in a gene locuscomprises an alteration that inhibits detection of a first autoinducerand wherein a second alteration in a gene locus comprises an alterationthat inhibits production of a second autoinducer and wherein the cell isbioluminescent when contacted with an autoinducer.

[0015] In another aspect, the invention provides a method foridentifying an autoinducer analog that regulates the activity of anautoinducer by contacting a bacterial cell, or extract thereof,comprising biosynthetic pathways which will produce a detectable amountof light in response to an autoinducer with an autoinducer analog andcomparing the amount of light produced by the bacterial cell, or extractthereof, in the presence of an autoinducer with the amount produced inthe presence of the autoinducer analog, wherein a change in theproduction of light is indicative of an autoinducer analog thatregulates the activity of an autoinducer.

[0016] In another aspect, the invention provides a method for producingautoinducer-2 by contacting S-adenosylhomocysteine (SAH) with a LuxSprotein under conditions and for such time as to promote the conversionof S-adenosylhomocysteine to autoinducer-2.

[0017] In another aspect, the invention provides a method for producingautoinducer-2 by contacting S-ribosylhomocysteine (SRH) with a LuxSprotein under conditions and for such time as to promote the conversionof S-ribosylhomocysteine to autoinducer-2.

[0018] In another aspect, the invention provides A method for producingautoinducer-2 by contacting S-adenosylhomocysteine (SAH) with a5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase protein underconditions and for such time as to promote the conversion ofS-adenosylhomocysteine to S-ribosylhomocysteine; contacting theabove-described S-ribosylhomocysteine with a LuxS protein underconditions and for such time as to promote the conversion ofS-ribosylhomocysteine to autoinducer-2.

[0019] In another aspect, the invention provides a method for detectingan autoinducer-associated bacterial biomarker by contacting at least onebacterial cell with an autoinducer molecule under conditions and forsuch time as to promote induction of a bacterial biomarker and detectingthe bacterial biomarker.

[0020] In another aspect, the invention provides a method for detectinga target compound that binds to a LuxP protein by contacting the LuxPprotein with the target compound and detecting binding of the compoundto LuxP.

[0021] In another aspect, the invention provides a method for regulatingbacterial biofilm formation comprising contacting a bacterium capable ofbiofilm formation with a compound capable of regulating biofilmformation, wherein the compound regulates autoinducer-2 activity.

[0022] According to another aspect of the invention, a method isprovided for purifying the aforementioned bacterial extracellularsignaling factor. The method comprises the steps of: (a) growing, in aculture medium, bacterial cells that produce the signaling molecule; (b)separating the bacterial cells from the culture medium; (c) incubatingthe bacterial cells in a solution having high osmolarity, underconditions that permit production and secretion of the signalingmolecule from the bacterial cells; (d) separating the bacterial cellsfrom the high osmolarity solution; and (e) purifying the factor from thehigh osmolarity solution. The method may further comprise: (f)separating polar factors from non-polar factors in an evaporated sampleof the high osmolarity solution; and (g) subjecting the polar factors toreverse-phase High Performance Liquid Chromatography. In a preferredembodiment, the high osmolarity solution comprises at least 0.4 Mmonovalent salt, most preferably 0.4-0.5 M NaCl. In another preferredembodiment, the method further comprises growing the bacterial cells ina culture medium containing a carbohydrate selected from the groupconsisting of glucose, fructose, mannose, glucitol, glucosamine,galactose and arabinose.

[0023] According to another aspect of the invention, an isolated nucleicacid molecule is provided, which encodes a protein necessary forbiosynthesis of a bacterial extracellular signaling factor that inducesexpression of a Vibrio harveyi LuxQ luminescence gene. The nucleic acidmolecule may be isolated from a wide variety of bacteria, including butnot limited to: Vibrio harveyi, Vibrio cholera, Salmonella typhimurium,Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillussubtilis and Borrelia burdorferi.

[0024] The aforementioned nucleic acid molecule encodes a protein havingbetween about 150 and 200 amino acid residues. Preferably, the encodedprotein comprises an amino acid sequence substantially the same as asequence selected from the group consisting of any of SEQ ID NOS:10-17,or a consensus sequence derived from a comparison of two or more of SEQID NOS: 10-17. The nucleic acid molecule preferably has a sequencesubstantially the same as a sequence selected from the group consistingof any of SEQ ID NOS:1-9, or a consensus sequence derived from acomparison of two or more of SEQ ID NOS: 1-9.

[0025] Recombinant DNA molecules comprising the aforementioned nucleicacid molecules are also provided in accordance with the presentinvention, as well as proteins produced by expression of any of thenucleic acid molecules.

[0026] Additional features and advantages of the present invention willbe better understood by reference to the drawings, detailed descriptionand examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1. Signaling substance from E. coli AB1157 and S. typhimuriumLT2 cell-free culture fluids that induces luminescence in V. harveyi.The responses of V. harveyi reporter strains BB170 (Sensor 1⁻, Sensor2⁺) (FIG. 1A), and BB886 (Sensor 1⁺, Sensor 2⁻) (FIG. 1B) to signalingsubstances present in cell-free culture fluids from E. coli, S.typhimurium and V. harveyi strains are shown. A bright culture of eachreporter strain was diluted 1:5000 into fresh medium, and the lightproduction per cell was then measured during the growth of the dilutedculture. Cell-free culture fluids or sterile growth medium were added ata final concentration of 10% (v/v) at the start of the experiment. Thedata for the 5 hour time point are shown and are presented as thepercent of the activity obtained when V. harveyi cell-free spent culturefluids are added. Abbreviations used for the different strains are: V.h;Vibrio harveyi, S.t; Salmonella typhimurium, and E.c; Escherichia coli.

[0028]FIG. 2. Active secretion of the signaling molecule by viable E.coli and S. typhimurium. The response of the V. harveyi reporter strainBB170 (Sensor 1⁻, Sensor 2⁺) to a signaling substance produced andsecreted by E. coli AB1157 and S. typhimurium LT2 but not E. coli DH5 isshown. V. harveyi reporter strain BB170 was diluted 1:5000 in AB mediumand light output per cell was monitored during growth. At the start ofthe experiment, either 1×10⁶ E. coli AB1157, S. typhimurium LT2 or E.coli DH5 washed and resuspended viable cells (left-hand, white bars) orUV-killed cells (right-hand, black bars) was added. The data arepresented as the fold-activation above the endogenous level ofluminescence expressed by V. harveyi BB170 at the 5 hour time point.Abbreviations used for the different strains are: S.t; Salmonellatyphimurium, and E.c; Escherichia coli.

[0029]FIG. 3. Effect of glucose depletion on the production anddegradation of the signaling activity by S. typhimurium LT2. S.typhimurium LT2 was grown in LB medium containing either 0.1% glucose(FIG. 3A) or 0.5% glucose (FIG. 3B). At the specified times cell-freeculture fluids were prepared and assayed for signaling activity in theluminescence stimulation assay (Bars), and the concentration of glucoseremaining (circles). The cell number was determined at each time bydiluting and plating the S. typhimurium LT2 on LB medium and countingcolonies the next day (squares). The signaling activity is presented asthe percent of the activity obtained when V. harveyi cell-free spentculture fluids are added. These data correspond to the 5 h time point inthe luminescence stimulation assay. The glucose concentration is shownas % glucose remaining. Cell number is cells/ml×10⁻⁹. The symbol \\indicates that the time axis is not drawn to scale after 8 h.

[0030]FIG. 4. Response curve of V. harveyi to AI-2 produced by V.harveyi and S. typhimurium. The V. harveyi reporter strain BB170 (Sensor1⁻, Sensor 2⁺) was tested for its response to the addition of exogenousAI-2 made by V. harveyi strain BB152 (AI-1⁻, AI-2⁺) and to that made byS. typhimurium LT2. A bright culture of the reporter strain was diluted1:5000 and either 10% (v/v) growth medium (closed circles), cell-freeculture fluid from V. harveyi BB152 grown overnight in AB (opencircles), or cell-free culture fluid from S. typhimurium LT2 grown for 6h on LB+0.5% glucose (closed squares) was added at the start of theexperiment. RLU denotes relative light units and is defined as (countsmin⁻¹×10³)/(colony-forming units ml⁻).

[0031]FIG. 5. Conditions affecting autoinducer production in S.typhimurium. S. typhimurium LT2 was subjected to a variety of treatmentsafter which cell-free culture fluids or osmotic shock fluids wereprepared. These preparations were added to a diluted culture of the V.harveyi AI-2 reporter strain BB170 at 10% (v/v) and light output wasmeasured thereafter. Fold activation is the level of light produced bythe reporter following addition of the specified S. typhimuriumpreparation divided by the light output of the reporter when growthmedium alone was added. The bars in FIG. 5A represent cell-free fluidsprepared from S. typhimurium after the following treatments: LB 6 h; 6 hgrowth in LB at 30° C., LB+Glc 6 h; 6 h growth in LB+0.5% glucose at 30°C., LB+Glc 24 h; 24 h growth in LB+0.5% glucose at 30° C. In all theexperiments presented in FIG. 5B, the S. typhimurium were pre-grown at30° C. for 6 h in LB containing 0.5% glucose, then pelleted andresuspended for 2 h under the following conditions: LB; in LB at 30° C.,LB+Glc; in LB+0.5% glucose at 30° C., LB pH 5; in LB at pH 5.0 at 30°C., 0.4M NaCl; in 0.4M NaCl at 30° C., 0.1M NaCl; in 0.1M NaCl at 30°C., and Heat Shock 430; in LB+0.5% glucose at 43° C. After these twohour treatments, cell-free fluids were prepared from each sample andassayed.

[0032]FIG. 6. S. typhimurium signaling activity in limiting andnon-limiting concentrations of glucose. S. typhimurium LT2 was grown inLB in the presence of limiting (0.1%) and non-limiting (1.0%)concentrations of glucose. The activity present in the cell-free culturefluids (black bars) was assayed at the times indicated and normalized tothat produced by 1×10⁹ cells. The increase in signaling activitymeasured in the 0.4M NaCl osmotic shock fluids prepared from the samecells is shown as the white bars on top of the black bars. These dataare also normalized for 1×10⁹ cells. The signaling activity for limitingglucose is shown in FIGS. 6A, 6C, and 6E, and that for non-limitingglucose is shown in FIGS. 6B, 6D, and 6F. FIGS. 6A and 6B also show thepercent glucose remaining (triangles), FIGS. 6C and 6D show the cellnumber (squares), and Panels E and F show the pH (circles) at each timepoint.

[0033]FIG. 7. Effects of glucose and pH on signal production by S.typhimurium. The quorum sensing signal released by S. typhimurium LT2was measured when the cells were grown in LB medium containing 0.5%glucose at pH 7.2 (FIG. 7A, bars), and when the cells were grown in LBat pH 5.0 without an added carbon source (FIG. 7B, bars). The level ofsignal present in cell free culture fluids (black bars) and in 0.4M NaClosmotic shock fluids was measured (white bars on top of black bars) atthe time points indicated. In each panel, the circles represent the pHof the medium, and the squares show the cell number at the differenttime points.

[0034]FIG. 8. High osmolarity induces signal release and low osmolarityinduces signal degradation in S. typhimurium LT2. The quorum sensingsignal released by S. typhimurium LT2 resuspended in 0.4M NaCl and in0.1M NaCl was measured in the presence and absence of protein synthesis.S. typhimurium LT2 was pre-grown in LB containing 0.5% glucose for 6 h.The cells were harvested and resuspended in 0.4 M NaCl (FIG. 8A) or 0.1M NaCl (FIG. 8B) in the presence or absence of 30 g/ml Cm for the timeperiods indicated. In each panel, the open symbols represent theactivity measured in the absence of Cm and the closed symbols representthe activity measured in the presence of Cm.

[0035]FIG. 9. The luxS and ygaG genes from V. harveyi and E. coliMG1655. FIG. 9A shows a restriction map of the V. harveyi luxS_(V.h.)chromosomal region which was defined by Tn5 insertion. The sites of Tn5insertions that disrupted the AI-2 production function and one controlTn5 insertion outside of the luxS_(V.h.) locus are shown (triangles).FIG. 9B depicts the ygaG region in the E. coli MG1655 chromosome. ThisORF is flanked by the emrB and gshA genes. The direction oftranscription of each gene is indicated by the horizontal arrows. Thecorresponding position of the MudJ insertion that eliminated AI-2production in S. typhimurium LT2 is shown by a vertical arrow. H, R, P,and B denote HindIII, EcoRI, PstI and BamHI restriction sitesrespectively.

[0036]FIG. 10. Autoinducer production phenotypes of V. harveyi and S.typhimurium strains. Cell-free culture fluids from V. harveyi and S.typhimurium strains were prepared and tested for AI-2 activity in the V.harveyi BB170 bioassay. FIG. 10A: AI-2 production phenotypes of the wildtype V. harveyi strain MM28 which contains a Tn5 insertion outside ofluxS_(V.h.) (denoted WT) and the luxS_(V.h.)::Tn5 mutant strain MM30(denoted luxS⁻). FIG. 10B: AI-2 production phenotypes of wild type S.typhimurium LT2 (denoted WT) and the ygaG::MudJ insertion mutant strainCS132 (denoted ygaG⁻). Activity is reported as fold-induction ofluminescence expression of the V. harveyi BB170 reporter strain overthat when sterile medium was added.

[0037]FIG. 11. Complementation of AI-2 production in S. typhimuriumCS132 and E. coli DH5. Cell-free culture fluids from E. coli and S.typhimurium strains were tested for AI-2 activity in the bioassay. Theactivity present in these fluids was compared to that produced by wildtype V. harveyi BB120. In the figure, the level of BB120 activity wasnormalized to 100%. FIG. 11A: AI-2 activity in cell-free fluids fromwild type V. harveyi BB120, E. coli O157:H7, and S. typhimurium LT2.FIG. 11B: Complementation of S. typhimurium CS132 (ygaG::MudJ) and FIG.11C: Complementation of E. coli DH5. In Panel B and C, the in trans AI-2production genes are the following: vector control (denoted: none), E.coli O157:H7 ygaG; and V. harveyi BB120 luxS_(V.h.) E. coli and V.harveyi are abbreviated E.c. and V.h. respectively.

[0038]FIG. 12. Alignment of LuxS and YgaG protein sequences. Thetranslated protein sequences for the AI-2 production family of proteinsare shown. We determined the sequences for the luxS_(V.h.) gene from V.harveyi BB120, and the ygaG genes (re-named herein as luxS_(E.c.) fromE. coli MG1655, E. coli O157:H7, and E. coli DH5. The S. typhimurium LT2ygaG (re-named herein luxS_(S.t.) partial sequence came from the S.typhimurium database. Amino acid residues that are not identical to theLuxS_(V.h.) protein are underlined and not in bold font. The site of theframe shift mutation in the E. coli DH5 DNA sequence is denoted by an“*”. The 20 altered amino acid residues that are translated followingthe frame shift are enclosed by the box.

[0039]FIG. 13. A diagram of the hybrid quorum sensing circuit of Vibrioharveyi is provided. The AI-1 and AI-2 circuits are independentlystimulated but integrate their signals for light expression. Eachpathway, however, is also independently competent to generate light.This allows for reciprocal mutations in the LuxN or LuxQ sensors to beused to construct a reporter specific for AI-2 or AI-1, respectively.

[0040]FIG. 14. Response phenotypes of V. harveyi wild-type and luxregulatory mutants. At the first time point, cell-free culture fluids(10%), or nothing (N.A) was added. Wild-type, cell-free culture fluid(AI-1+AI2); LuxS-cell-free culture fluid (AI-1); LuxM-cell-free culturefluid (AI-2). Relative light units are defined as cpm×10³/CFU/ml.

[0041]FIG. 15. A diagram of the biosynthetic pathway of autoinducer-2(AI-2) is shown.

[0042]FIG. 16. The structure of AI-2 and the biosynthetic precursorsfrom which AI-2 is derived are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0043] In accordance with the present invention, we have identified,isolated and characterized an extracellular signaling factor produced byseveral strains of pathogenic bacteria, including Salmonella typhimuriumand Escherichia coli, which has a role in regulating the pathogenesis orvirulence of these bacteria. We have also identified and cloned genesinvolved in the biosynthesis of this signaling factor. The purificationand/or cloning of this signaling molecule and the genes that encodeproteins that catalyze its biosynthesis open a new avenue for drugdesign aimed at either inhibition of production of or response to thismolecule by bacteria. Drugs designed to interfere with signaling by thismolecule will constitute a new class of antibiotics. The inventionfurther provides methods for detecting an autoinducer and methods forthe in vitro production of autoinducr-2.

[0044] I. Definitions:

[0045] Various terms relating to the biological molecules of the presentinvention are used throughout the specifications and claims. The terms“substantially the same,” “percent similarity” and “percent identity”are defined in detail below.

[0046] With reference to the novel signaling factor of the presentinvention, this molecule is alternatively referred to herein as“signaling factor”, “signaling molecule”, “autoinducer”, and morespecifically, “autoinducer-2” or “AI-2”. The terms “autoinducer-2” and“AI-2” refer specifically to the signaling factor as produced by Vibrioharveyi. The terms “signaling factor” or “signaling molecule”,“autoinducer” or “AI-2-like molecule” are intended to refer generally tothe signaling factors of the present invention, of which AI-2 is anexample.

[0047] With reference to nucleic acids of the invention, the term“isolated nucleic acid” is sometimes used. This term, when applied toDNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous (in the 5′ and 3′ directions) in thenaturally occurring genome of the organism from which it was derived.For example, the “isolated nucleic acid” may comprise a DNA moleculeinserted into a vector, such as a plasmid or virus vector, or integratedinto the genomic DNA of a procaryote or eucaryote. An “isolated nucleicacid molecule” may also comprise a cDNA molecule.

[0048] With respect to RNA molecules of the invention, the term“isolated nucleic acid” primarily refers to an RNA molecule encoded byan isolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been sufficiently separated from RNAmolecules with which it would be associated in its natural state (i.e.,in cells or tissues), such that it exists in a “substantially pure” form(the term “substantially pure” is defined below).

[0049] With respect to protein, the term “isolated protein” or “isolatedand purified protein” is sometimes used herein. This term refersprimarily to a protein produced by expression of an isolated nucleicacid molecule of the invention. Alternatively, this term may refer to aprotein which has been sufficiently separated from other proteins withwhich it would naturally be associated, so as to exist in “substantiallypure” form.

[0050] The term “substantially pure” refers to a preparation comprisingat least 50-60% by weight the factor of interest (e.g., pathogenesissignaling factor, nucleic acid, oligonucleotide, protein, etc.). Morepreferably, the preparation comprises at least 75% by weight, and mostpreferably 90-99% by weight, the factor of interest. Purity is measuredby methods appropriate for the factor of interest (e.g. chromatographicmethods, agarose or polyacrylamide gel electrophoresis, HPLC analysis,and the like).

[0051] With respect to antibodies of the invention, the term“immunologically specific” refers to antibodies that bind to one or moreepitopes of a protein of interest, but which do not substantiallyrecognize and bind other molecules in a sample containing a mixedpopulation of antigenic biological molecules.

[0052] With respect to oligonucleotides, the term “specificallyhybridizing” refers to the association between two single-strandednucleotide molecules of sufficiently complementary sequence to permitsuch hybridization under pre-determined conditions generally used in theart (sometimes termed “substantially complementary”). In particular, theterm refers to hybridization of an oligonucleotide with a substantiallycomplementary sequence contained within a single-stranded DNA or RNAmolecule of the invention, to the substantial exclusion of hybridizationof the oligonucleotide with single-stranded nucleic acids ofnon-complementary sequence.

[0053] The term “promoter region” refers to the transcriptionalregulatory regions of a gene, which may be found at the 5′ or 3′ side ofthe coding region, or within the coding region, or within introns.

[0054] The term “selectable marker gene” refers to a gene encoding aproduct that, when expressed, confers a selectable phenotype such asantibiotic resistance on a transformed cell.

[0055] The term “reporter gene” refers to a gene that encodes a productwhich is easily detectable by standard methods, either directly orindirectly.

[0056] The term “operably linked” means that the regulatory sequencesnecessary for expression of the coding sequence are placed in the DNAmolecule in the appropriate positions relative to the coding sequence soas to enable expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and otherregulatory elements (e.g., enhancers or translation regulatorysequences) in an expression vector.

[0057] II. Description of the Signaling Factor

[0058] The invention provides a heterologous bio-assay that has enabledthe identification of an extracellular signaling factor produced by S.typhimurium and E. coli, among other pathogenic bacteria. The factor issometimes referred to herein as a “pathogenesis signaling” factor ormolecule, though it acts as a signal for a variety of physiologicalchanges in bacteria other than pathogenesis. The factor mimics theaction of AI-2 (autoinducer-2) of the quorum sensing bacterium Vibrioharveyi, and it acts specifically through the V. harveyi SignalingSystem 2 detector, LuxQ.

[0059] The signaling factor is a small, soluble, heat labile organicmolecule that is involved in intercellular communication in all threebacteria. In E. coli and Salmonella, maximal secretion of the moleculeoccurs in mid-exponential phase and the extracellular activity isdegraded as glucose becomes depleted from the medium or by the onset ofstationary phase. Destruction of the signaling molecule in stationaryphase indicates that, in contrast to other quorum sensing systems,quorum sensing in bacteria that utilize the signaling molecule iscritical for regulating behavior in the pre-stationary phase of growth.Protein synthesis is required for degradation of the activity,indicating that a complex regulatory circuitry controls quorum sensingin these enteric bacteria.

[0060] Increased signaling activity is observed if, after growing in thepresence of glucose, the bacteria are transferred to a high osmolarity(e.g., 0.4M NaCl) or to a low pH (e.g., pH 5.0) environment. Moreover,degradation of the signal is induced by conditions of low osmolarity(e.g., 0.1M NaCl. High osmolarity and low pH are two conditionsencountered by pathogenic enteric bacteria, such as S. typhimurium andE. coli, when they undergo the transition to a pathogenic existenceinside a host organism. Thus, quorum sensing in these bacteria appearsto play a role in regulating their virulence, by way of directing thebacteria to undergo the transition between a host-associated (i.e.,pathogenic) and a free-living existence.

[0061] Other factors that regulate the activity of the signalingmolecule are described in greater detail in

EXAMPLE 2 Particularly Exemplified is the Regulation of the Molecule inS. typhimurium

[0062] The timing of lux induction in the bio-assay and the shape of theresponse curve of V. harveyi to the S. typhimurium and E. coli signalsare indistinguishable from those of V. harveyi responding to its ownSignaling System 2 inducer, AI-2. Furthermore, each of the signalingmolecules from S. typhimurium, E. coli and V. harveyi can be partiallypurified according to the same protocol. These results indicate that thesignaling molecules from each of the aforementioned organisms are eitheridentical or very closely related. Accordingly, AI-2 from V. harveyi isa signaling molecule of the invention, but appears to play a differentrole in that organism than it does in pathogenic enteric bacteria suchas Salmonella and Escherichia.

[0063] A. Structure of the AI-2 Signaling Factor

[0064] Thus, in another aspect, the invention provides autoinducer-2(AI-2) signaling factor and derivatives thereof. AI-2 of the inventioncan be used to regulate bacterial growth in a variety of applications.The present invention provides autoinducer-2 molecules having thestructure:

[0065] wherein R1, R2, R3 and R4 are independently selected fromhydrido, halo, alkyl, haloalkyl, cycloalkyl, cycloalkenyl, heterocyclyl,methyl, cyano, alkoxycarbonyl, amino, carboxyl, hydroxyl, formyl, nitro,fluoro, chloro, bromo, methyl, aryl, heteroaryl, aralkyl,heteroarylalkyl, alkylsulfonyl, haloalkylsulfonyl, arylsulfonyl,heteroarylsulfonyl, hydroxyalkyl, mercaptoalkyl, alkoxyalkyl,aryloxyalkyl, heteroaryloxyalkyl, aralkyloxyalkyl,heteroarylalkyloxyalkyl, alkylthioalkyl, arylthioalkyl,heteroarylthioalkyl, aralkylthioalkyl, heteroarylalkylthioalkyl,haloalkylcarbonyl, haloalkyl(hydroxy)alkyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl,carboxyalkyl, alkoxycarbonylalkyl, alkylcarbonyloxyalkyl, aminoalkyl,alkylaminoalkyl, arylaminoalkyl, aralkylaminoalkyl,heteroarylaminoalkyl, heteroarylalkylaminoalkyl, alkoxy, and aryloxy;phenyl, cyclohexyl, cyclohexenyl, benzofuryl, benzodioxolyl, furyl,imidazolyl, thienyl, thiazolyl, pyrrolyl, oxazolyl, isoxazolyl,triazolyl, pyrimidinyl, isoquinolyl, quinolinyl, benzimidazolyl,indolyl, pyrazolyl and pyridyl, aminosulfonyl, fluoro, chloro, bromo,methylthio, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl,hexyl, cyano, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,tert-butoxycarbonyl, propoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,pentoxycarbonyl, methylcarbonyl, fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl,dichloropropyl, methoxy, methylenedioxy, ethoxy, propoxy, n-butoxy,hydroxymethyl, hydroxyethyl, methoxymethyl, ethoxymethyl,trifluoromethoxy, methylamino, N,N-dimethylamino, phenylamino,ethoxycarbonylethyl, and methoxycarbonylmethyl, methyl, ethyl,fluoromethyl, difluoromethyl, trifluoromethyl, cyano, methoxycarbonyl,ethoxycarbonyl, tert-butoxycarbonyl, benzyl, phenylethyl, phenylpropyl,methylsulfonyl, phenylsulfonyl, trifluoromethylsulfonyl, hydroxymethyl,hydroxyethyl, methoxymethyl, ethoxymethyl, methylcarbonyl,ethylcarbonyl, trifluoromethylcarbonyl, trifluoro(hydroxy)ethyl,phenylcarbonyl, benzylcarbonyl, methoxycarbonylmethyl,ethoxycarbonylethyl, carboxymethyl, carboxypropyl,methylcarbonyloxymethyl, phenyloxy, phenyloxymethyl, thienyl, furyl, andpyridyl, wherein the thienyl, furyl, pyridyl, methylthio,methylsulfinyl, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl,hexyl, cyano, fluoromethyl, difluoromethyl, trifluoromethyl,chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl,heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl,difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, methoxy,methylenedioxy, ethoxy, propoxy, n-butoxy, hydroxymethyl, hydroxyethyland trifluoromethoxy.

[0066] The chemical groups disclosed herein are known to those of skillin the art. For example, as used herein, the term “hydrido” denotes asingle hydrogen atom (H). This hydrido radical may be attached, forexample, to an oxygen atom to form a hydroxyl radical or two hydridoradicals may be attached to a carbon atom to form a methylene (—CH2-)radical. In addition, alkyl radicals are “lower alkyl” radicals havingone to about ten carbon atoms. Most preferred are lower alkyl radicalshaving one to about six carbon atoms. Examples of such radicals includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, pentyl, iso-amyl, hexyl and the like. The term “halo” meanshalogens such as fluorine, chlorine, bromine or iodine. The terms“carboxy” or “carboxyl” denotes —CO2H. The term “carbonyl”, whether usedalone or with other terms, denotes —(S═O)—.

[0067] Preferably, the autoinducer-2 molecule of the invention is4,5-Dihidroxy-2,3-pentanedione having the structure:

[0068] As used herein, an “autoinducer-2 (AI-2)” molecule of theinvention includes a molecule that acts as a diffusable sensor forquorum sensing Signaling System 2. For example, AI-2 can regulate geneexpression by increasing or decreasing expression of genes associatedwith pathogenesis of a microorganism. Typically, autoinducer moleculesare produced by microorganisms, such as bacteria, during metabolism. Forexample, the autoinducer-2 (AI-2) molecule of the invention can interactwith LuxP which is the protein encoded by the homologue of the luxP geneof pathogenic bacteria such as V. cholerae, S. typhimurium and E. coli.In turn, the AI-2-LuxP complex can interact with LuxQ which is theprotein product encoded by the luxQ gene. The AI-2-LuxP-LuxQ interactioncan promote luminescence in bacteria such as Vibrio spp. TheAI-2-LuxP-LuxQ interaction has been linked to the activation ofbiochemical pathways required for bacterial pathogenicity. Thus, theinvention provides a method for controlling bacterial gene expressionand for regulating bacterial pathogenicity by modulating AI-2-LuxP-LuxQinteractions.

[0069] In another aspect, the invention provides methods for usinghomocysteine as an autoinducer molecule. The structure of homocysteineis as follows:

[0070] Homocysteine is produced by the activity of the LuxS protein onS-ribosylhomocysteine (FIG. 16). Thus, the invention provides methodsfor using homoserine as an autoinducer.

[0071] The present invention also encompasses optically active isomersof an autoinducer-2 molecule. As used herein, an “isomer” is intended toinclude molecules having the same molecular formula as an autoinducer-2molecule of the invention but possessing different chemical and physicalproperties due to a different arrangement of the atoms in the molecule.Isomers include both optical isomers and structural isomers. As usedherein, “optically active” is intended to include molecules that havethe ability to rotate a plane of polarized light. An optically activeisomer includes the L-isomer and the D-isomer of an autoinducer-2molecule of the invention.

[0072] In addition to optically active isomers, analogs of anautoinducer-2 molecule are included in the invention. As used herein, anAI-2 “analog” is intended to include molecules that are structurallysimilar but not identical to the claimed autoinducer molecule4,5-Dihidroxy-2,3-pentanedione. Analogs of AI-2 can include moleculesthat inhibit rather than stimulate the activity of the LuxP protein. Forexample, an analog of AI-2 that is capable of a nonproductiveinteraction with LuxP can be produced. Such a molecule can retain theability to bind to LuxP, but the analog AI-2-LuxP complex will not beable to productively interact with LuxQ resulting in an inhibition ofbacterial pathogenicity. Thus, an AI-2 analog of the invention can actas an inhibitor of bacterial pathogenesis by competing with endogenousAI-2 for binding to LuxP. In addition, an analog of AI-2 can beconstructed such the analog AI-2-LuxP complex is capable ofnonproductively interacting with LuxQ. In this case, the analogAI-2-LuxP-LuxQ complex is rendered nonfunctional for subsequentbiochemical processes such as, for example, transcriptional activationof genes required for pathogenicity. The invention also includes AI-2analogs which act synergistically to enhance the ability of AI-2 toincrease the activity of the LuxP protein.

[0073] B. Preparation of the Signaling Factor

[0074] Initial strategies for purifying the signaling molecule of theinvention resulted in a partially purified preparation comprising themolecule with a specific signaling activity estimated at about 0.1-1.0mg of the partially purified material stimulating a 1,000-fold increasein luminescence, as measured in the V. harveyi bioassay. The signalingactivity does not extract quantitatively into organic solvents and itdoes not bind to either a cation or an anion exchange column. Themolecule is a small (less than 1,000 kDa), polar but uncharged organicfactor. The activity is acid stable and base labile, and it is heatresistant to 80° C. but not 100° C. These features of the signalingmolecule make it clear that the molecule is different from anypreviously described autoinducer.

[0075] The signaling factor of the present invention may be purifiedfrom its natural sources, i.e. the bacteria that produce it. With regardto purifying AI-2 from natural sources, altering the culture medium,e.g., by adding glucose or another sugar, by increasing the osmolarity,and/or decreasing pH, can increase production of the signaling moleculein Salmonella and other enteric bacteria, has also enabled purificationof the signaling molecule to near-homogeneity. Thus, the molecule hasnow been highly purified from culture fluids of enteric bacteria (e.g.,E. coli, S. typhimurium) using the following protocol:

[0076] 1. Grow a culture of the signal producing enteric bacteriumovernight in LB containing 0.5% glucose or another sugar (37° C., withaeration). Inoculate fresh LB containing glucose or another sugar at0.5% with the overnight culture, at a 1:100 dilution. Grow the dilutedculture to mid-exponential phase (3.5 h, 37° C., with aeration).

[0077] 2. Pellet the cells (10,000 rpm, 10 min, 4° C.). Discard theculture medium. Resuspend the cells and wash in {fraction (1/10)} th theoriginal volume of low osmolarity NaCl solution (0.1M NaCl in water).

[0078] 3. Pellet the cells again (10,000 rpm, 10 min, 4° C.). Discardthe low osmolarity culture fluid. Resuspend the cells in {fraction(1/10)} th the original volume of high osmolarity NaCl solution (0.4MNaCl in water). Incubate the suspension at 37° C. for 2 h with aeration.During this time, increased production and secretion of the signalingmolecule occurs.

[0079] 4. Pellet the cells (10,000 rpm, 10 min, 4° C.). Collect thesupernatant containing the secreted signaling molecule, filter thesupernatant through a 0.2 M bacterial filter to remove any remainingcells.

[0080] 5. Evaporate the aqueous filtrate using a rotary evaporator at30° C. Extract the dried filtrate in {fraction (1/10)} th the originalvolume of chloroform:methanol (70:30).

[0081] 6. Evaporate the organic extract using a rotary evaporator atroom temperature. Re-dissolve the dried extract in methanol at {fraction(1/100)} th of the original volume.

[0082] 7. Subject the partially purified signal to High PerformanceLiquid Chromatography (HPLC), using a preparative reverse phase C18column. Elute the molecule with a linear gradient of 0-100% acetonitrilein water at 5 ml per minute. Collect 30 fractions, 5 ml each.

[0083] 8. Assay the HPLC fractions in the V. harveyi BB170 AI-2 assay,and pool the active fractions.

[0084] The product from the C18 column contains the signaling moleculeand a small number of other organic molecules. This highly purifiedpreparation of the signaling molecule has activity 50-100 times greaterthan that of the partially purified material described above (thepreparation of which did not include the high osmoticum step or thefinal HPLC step), i.e., 1-10 μg material stimulates a 1,000-foldincrease in luminescence in the V. harveyi bioassay.

[0085] Subsequent strategies for purifying the AI-2 signaling moleculehave led to the identification of a novel in vitro system for producingAI-2. Thus, in addition to providing a cloned, overexpressed andpurified S. typhimurium LuxS protein, the present invention alsoprovides a method for producing AI-2 in vitro. The present inventionprovides a mechanism for generating large quantities of pure AI-2 usefulfor mass spectral and NMR analysis, and for screening compounds whichregulate the activity of AI-2. Moreover, the present invention providesa method for determining the in vivo biosynthetic pathway for AI-2synthesis. The in vitro method for AI-2 production is described below inExample 5 and FIG. 15. The method provides a novel means for efficientlyproducing autoinducer molecules for further study. The method alsoprovides a means for producing substantial quantities of AI-2 for use incommercial applications. Such applications include, but are not limitedto, adding AI-2 of the invention to a growth media to increase bacterialgrowth. Such a method is particularly useful in the in the production ofantibiotics from cultured bacteria. The addition of AI-2 can increasethe antibiotic production of such organisms by promoting cell growth.Preferably, the signaling factor AI-2 is produced by the in vitro methodset forth in Example 5 of the disclosure.

[0086] C. Uses of the Signaling Factor

[0087] The isolated and purified signaling molecules of the presentinvention are used as targets for the design of compounds that regulatethe activity of AI-2. As used herein, “regulate” includes increasing ordecreasing the activity of AI-2. As used herein, the “activity” of AI-2encompasses any aspect of the molecules ability to act as a signalingfactor in bacterial quorum sensing. A “compound” can be any agent orcomposition that effects the activity of AI-2. For example, a compoundof the invention can be a nucleic acid, a protein or small molecule.Thus, the invention provides a means for identifying a new class ofantibiotics that inhibit the activity of the AI-2 molecule or otherwiseblock the signaling pathway in which the molecule participates. Suchinhibitors may be identified by large-scale screening of a variety oftest compounds, using the V. harveyi bioassay in the presence of thepurified signaling molecule. A reduction in signaling activity in thepresence of a test compound would be indicative of the ability of thatcompound to inhibit the activity of the signaling molecule or to blocksome other part of the pathogenesis signaling pathway.

[0088] Further, the invention provides a basis for the rational designof specific inhibitors or non-functional analogs of AI-2. Suchstructure-specific inhibitors or analogs may be tested in the V. harveyibioassay for their ability to inhibit the signaling molecule or to blockthe pathogenesis signaling pathway.

[0089] The invention also encompasses methods for identifying naturallyproduced compounds that inhibit the activity of a signaling moleculesuch as autoinducer-2. For example, a defensive strategy employed byeucaryotic organisms to avoid bacterial colonization is to specificallytarget and inhibit quorum sensing controlled functions. Such a mechanismhas been identified in D. pulchra. Recent studies indicate thathalogenated furanones produced by D. pulchra inhibit quorum sensing bycompeting for the homoserine-lactone (HSL) autoinducer-binding site inLuxR. Thus, by providing a novel autoinducer and the cellular componentsthat interact with the autoinducer, the present invention also providesa method to screen naturally produced compounds for their effect onquorum sensing system-2. For example, naturally produced compounds canbe screened for their effect on the autoinducer-2-LuxP interaction.Alternatively, such compounds can be screened for their effect onautoinducer-2-LuxP-LuxQ interactions.

[0090] It will be appreciated by persons skilled in the art that, nowthat targets for the signaling molecule have been identified in E. coli,inhibition of the E. coli target can also be used to screen potentialsignaling molecule inhibitors or analogs. The inventors have prepared aler-lacZ reporter fusion construct to be used in testing for reductionof expression of the Type III secretion gene in E. coli O157:H7(pathogenic strain) directly. Furthermore, a similar locus exists in S.typhimurium.

[0091] Thus, the invention provides a method for selecting inhibitors orsynergists of the autoinducer-2 molecule,4,5-Dihidroxy-2,3-pentanedione. As used herein, an “inhibitor” of AI-2is intended to include molecules that interfere with the ability of theautoinducer molecule to act as a signal for luminescence orpathogenesis. Inhibitors include molecules that degrade or bind to AI-2.The method comprises contacting the autoinducer molecule with asuspected inhibitor or synergist, measuring the ability of the treatedautoinducer molecule to stimulate the activity of a selected gene thendetermining whether the suspected inhibitor or synergist represses orenhances the activity of the autoinducer molecule. Actual inhibitors andsynergists of the autoinducer molecule are then selected. For example, asuspected inhibitor can be mixed with 4,5-Dihidroxy-2,3-pentanedione andthe mixture then combined with a reporter strain of V. harveyi disclosedherein. The amount of luminescence in the presence of the suspectedinhibitor can be compared with a control mixture which does not includethe inhibitor. A decrease in luminescence is indicative of AI-2inhibition. In this manner, compounds that regulate bacterialpathogenesis can be rapidly screened.

[0092] In another aspect, the invention also provides methods ofselecting inhibitory and synergistic analogs of AI-2. The methodcomprises mixing a known amount of the autoinducer molecule with a knownamount of the suspected inhibitory or synergistic analog, measuring theability of the treated autoinducer molecule to stimulate the activity ofa selected gene then determining whether the suspected inhibitory orsynergistic analog represses or enhances the activity of the autoinducermolecule. Actual inhibitory or synergistic analogs of the autoinducermolecule are then selected.

[0093] The autoinducer-2 molecule can be purified from the native sourceusing conventional purification techniques, derived synthetically bychemical means, or preferably, produced by the in vitro method of theinvention described below. As used herein, “purified from a nativesource” is intended to include an autoinducer-2 molecule of the aboveformula that has been manufactured by an organism. “Purified from thenative source” includes isolating the autoinducer molecule from theculture media or cytoplasm of bacteria such as S. typhimurium usingconventional purification techniques. As used herein, “synthesized bychemical means” is intended to include autoinducer molecules of theclaimed formula that have been artificially produced outside of anorganism. The invention includes an autoinducer of the inventionmanufactured by a person skilled in the art from chemical precursorsusing standard chemical synthesis techniques.

[0094] The invention further provides methods of inhibiting theinfectivity of a pathogenic organism as well as therapeutic compositionscontaining an AI-2 analog or AI-2 inhibitor of the invention. Themethods comprise administering to a subject a therapeutically effectiveamount of an pharmaceutical composition that is capable of inhibitingthe activity of AI-2. As used herein, “inhibiting infectivity” includesmethods of affecting the ability of a pathogenic organism to initiallyinfect or further infect a subject that would benefit from suchtreatment. A pharmaceutical composition of the invention can include,but is not restricted to, an agent that prevents the transcriptionalactivation of extracellular virulence factors such as exotoxin A andelastolytic proteases. As used herein, an “agent” includes moleculesthat inhibit the ability of the LuxP protein and LuxQ protein toactivate transcription of extracellular virulence factors. Agentsinclude inhibitors that interact directly with AI-2 such that AI-2 isprevented from acting as a sensor for quorum sensing Signaling System-2.Preferably, the agent interacts with 4,5-Dihidroxy-2,3-pentanedione.Agents further include analogs of AI-2 that can compete with4,5-Dihidroxy-2,3-pentanedione for binding to LuxP or LuxQ.

[0095] The invention further provides pharmaceutical compositions forpreventing or treating pathogen-associated diseases by targeting factorsinvolved in the Signaling System type-2 pathway. For example, LuxP orLuxQ, or homologues thereof, provide a common target for the developmentof a vaccine. Antibodies raised to LuxP or LuxQ, or homologues thereof,can inhibit the activation of bacterial pathways associated withvirulence. Thus, LuxP and LuxQ provide common antigenic determinantswhich can be used to immunize a subject against multiplepathogen-associated disease states. For example, the autoinducerSignaling System type-2 is believed to exist in a broad range ofbacterial species including bacterial pathogens. As discussed above, theautoinducer-2 signaling factor is believed to be involved ininter-species as well as intra-species communication. In order for thequorum sensing Signaling System type-2 to be effective for inter-speciescommunication, it is likely to be highly conserved among variousbacterial species. Thus, challenging a subject with the LuxP or LuxQpolypeptide, or an antigenic fragment thereof, isolated from aparticular organism may confer protective immunity to other diseasestates associated with a different organism. For example, a vaccinedeveloped to the LuxP protein isolated from V. cholerae may be capableof cross-reacting with a LuxP homologue expressed by a differentorganism. Thus, it is envisioned that methods of the present inventioncan be used to treat pathogen-associated disease states.

[0096] Generally, the terms “treating”, “treatment”, and the like areused herein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a spirochete infection or disease or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure for an infection or disease and/or adverse effectattributable to the infection or disease. “Treating” as used hereincovers any treatment of (e.g., complete or partial), or prevention of,an infection or disease in a mammal, particularly a human, and includes:

[0097] (a) preventing the disease from occurring in a subject that maybe predisposed to the disease, but has not yet been diagnosed as havingit;

[0098] (b) inhibiting the infection or disease, i.e., arresting itsdevelopment; or

[0099] (c) relieving or ameliorating the infection or disease, i.e.,cause regression of the infection or disease.

[0100] Thus, the invention includes various pharmaceutical compositionsuseful for ameliorating symptoms attributable to a bacterial infectionor, alternatively, for inducing a protective immune response to preventsuch an infection. For example, a pharmaceutical composition accordingto the invention can be prepared to include an antibody against, forexample, LuxP or LuxQ, a peptide or peptide derivative of LuxP or LuxQ,a LuxP or LuxQ mimetic, or a LuxP or LuxQ-binding agent according to thepresent invention into a form suitable for administration to a subjectusing carriers, excipients and additives or auxiliaries. Frequently usedcarriers or auxiliaries include magnesium carbonate, titanium dioxide,lactose, mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

[0101] The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Langer, Science, 249:1527, (1990); Gilman et al. (eds.) (1990), each of which is hereinincorporated by reference.

[0102] As used herein, “administering a therapeutically effectiveamount” is intended to include methods of giving or applying apharmaceutical composition of the invention to a subject which allow thecomposition to perform its intended therapeutic function. Thetherapeutically effective amounts will vary according to factors such asthe degree of infection in a subject, the age, sex, and weight of theindividual. Dosage regima can be adjusted to provide the optimumtherapeutic response. For example, several divided doses can beadministered daily or the dose can be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0103] The pharmaceutical composition can be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids and othernatural conditions which may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

[0104] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

[0105] Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated above.

[0106] The pharmaceutical composition can be orally administered, forexample, with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the pharmaceutical composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5 toabout 80% of the weight of the unit. The amount of pharmaceuticalcomposition in such therapeutically useful compositions is such that asuitable dosage will be obtained.

[0107] The tablets, troches, pills, capsules and the like can alsocontain the following: a binder such as gum gragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier. Various other materials can be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules can be coated with shellac,sugar or both. A syrup or elixir can contain the agent, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, thepharmaceutical composition can be incorporated into sustained-releasepreparations and formulations.

[0108] As usd herein, a “pharmaceutically acceptable carrier” isintended to include solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the pharmaceutical composition, usethereof in the therapeutic compositions and methods of treatment iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

[0109] It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the individualto be treated; each unit containing a predetermined quantity ofpharmaceutical composition is calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the novel dosage unit forms of theinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the pharmaceutical composition and the particulartherapeutic effect to be achieve, and (b) the limitations inherent inthe art of compounding such an pharmaceutical composition for thetreatment of a pathogenic infection in a subject.

[0110] The principal pharmaceutical composition is compounded forconvenient and effective administration in effective amounts with asuitable pharmaceutically acceptable carrier in an acceptable dosageunit. In the case of compositions containing supplementary activeingredients, the dosages are determined by reference to the usual doseand manner of administration of the said ingredients.

[0111] In addition to generating antibodies which bind to antigenicepitopes of proteins of the invention, it is further envisioned that themethod of the invention can be used to induce cellular responses,particularly cytotoxic T-lymphocytes (CTLs), to antigenic epitopes of,for example LuxP or LuxQ. Typically, unmodified soluble proteins fail toprime major histocompatibility complex (MHC) class I-restricted CTLresponses whereas particulate proteins are extremely immunogenic andhave been shown to prime CTL responses in vivo. CTL epitopes and helperepitopes have been identified in proteins from many infectiouspathogens. Further, these epitopes can be produced concurrently suchthat multiple epitopes can be delivered in a form that can prime MHCclass I restricted CTL responses. An example of a system that canproduce recombinant protein particles carrying one or more epitopesentails the use of the p1 protein of the retrotransposon Ty1 ofSaccharomyces cerevisiae (Adams, et al., Nature, 329:68, 1987).Sequences encoding CTL epitopes can, for example, be fused to theC-terminus of p1 and the resulting Ty virus-like particles (Ty-VLPS) maybe able to generate a CTL response. Thus, conserved regions ofpathogenic antigens, such as those that are involved in, or result from,the activation of Signaling System type-2, can be identified andincorporated together in a particle which enables the host immune systemto mount an effective immune response against multiple spirochetalorganisms. Further, the method of the invention can be used to generateparticles with multiple epitopes to a single protein, such as LuxP, ormultiple epitopes from various proteins.

[0112] The method of the invention also includes slow release antigendelivery systems such as microencapsulation of antigens into liposomes.Such systems have been used as an approach to enhance the immunogenicityof proteins without the use of traditional adjuvants. Liposomes in theblood stream are generally taken up by the liver and spleen, and areeasily phagocytosed by macrophages. Liposomes also allow co-entrapmentof immunomodulatory molecules along with the antigens, so that suchmolecules may be delivered to the site of antigen encounter, allowingmodulation of the immune system towards protective responses.

[0113] In another embodiment, the invention provides a method foridentifying a compound which binds to a protein of the invention, suchas LuxP or LuxQ. The method includes incubating components comprisingthe compound and LuxP or LuxQ under conditions sufficient to allow thecomponents to interact and measuring the binding of the compound to LuxPor LuxQ. Compounds that bind to LuxP or LuxQ include peptides,peptidomimetics, polypeptides, chemical compounds and biologic agents asdescribed above.

[0114] Incubating includes conditions which allow contact between thetest compound and LuxP or LuxQ. Contacting includes in solution and insolid phase. The test ligand(s)/compound may optionally be acombinatorial library for screening a plurality of compounds. Compoundsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs)(Landegren, et al., Science, 241:1077, 1988), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren, et al.,Science, 242:229-237, 1988). Also included in the screening method ofthe invention are combinatorial chemistry methods for identifyingchemical compounds that bind to LuxP or LuxQ. See, for example, Plunkettand Ellman, “Combinatorial Chemistry and New Drugs”, ScientificAmerican, April, p.69, (1997).

[0115] The invention further provides a method for promoting theproduction of a bacterial product, such as, for example, an antibiotic,by contacting a culture of bacteria with an AI-2 of the invention at aconcentration effective to stimulate or promote cellular metabolism,growth or recovery. For example, it is known that antibiotic-producingbacteria only produce an antibiotic at or near the peak of log phasegrowth. By contacting a culture medium containing suchantibiotic-producing bacteria with AI-2 of the invention, production ofan antibiotic can be induced at an earlier phase of growth. Thus, AI-2of the invention provides a method for increasing the amount ofantibiotic produced by a culture. “Culture medium”, as used herein, isintended to include a substance on which or in which cells grow. Theautoinducer molecule can be included in commercially available cellculture media including broths, agar, and gelatin.

[0116] The invention further provides a method for identifying factorsthat degrade or inhibit synthesis autoinducer-2. For example, it isknown that autoinducer-1 concentration peaks in mid- to late log phaseof a bacterial cell culture. In contrast, autoinducer-2 concentrationincreases earlier in log phase of bacterial cell culture growth and ispresent in lower amounts in late log phase and stationary phase. Thisdata indicates that a mechanism exists for the degradation ofautoinducer-2 at a specific point in bacterial growth. By providingisolated and purified autoinducer-2, the invention allows for theidentification of the mechanism whereby autoinducer-2 levels arecontrolled. For example, partially purified bacterial extracts can beassayed against isolated autoinducer-2 to identify those fractions whichdegrade autoinducer-2. Fractions that degrade autoinducer-2 can befurther fractionated by techniques known to those skilled in the artuntil those cellular components involved in autoinducer degradation areisolated.

[0117] The present invention also provides a method of regulating theexpression of a gene. The method comprises inserting a gene intobacteria chosen for enhancement of gene expression by an agent capableof stimulating the activity of the LuxQ protein and incubating thebacteria with an agent capable of stimulating the activity of the LuxPprotein. Thus, the signaling molecule of the invention can also be usedin screens for other targets that are regulated by the molecule. Clonedpromoter-fusion libraries can be prepared from any species of bacteriaand these libraries can be used to identify genes that are induced orrepressed by the signaling factor, simply by screening for differencesin reporter activity in petri or microtiter plates containing thesignaling molecule compared to plates that do not contain the molecule.

[0118] In addition, quorum sensing is a major regulator of biofilmcontrol and quorum sensing blockers can therefore be used to preventand/or inhibit biofilm formation. Also, quorum sensing blockers areeffective in removing, or substantially decreasing the amount of,biofilms which have already formed on a surface. Thus, by providing thestructure of autoinducer-2 (AI-2), the present invention provides a newapproach to identifying compounds which inhibit bacterial infections byregulating biofilm formation.

[0119] It is known that quorum sensing blockers can reduce proteaseproduction by 50% in some strains of bacteria but the discovery thatcertain compounds can substantially eliminate protease productionimparts clear significant clinical advantages. Furthermore, theunexpected finding that biofilm formation can be inhibited or preventedby quorum sensing blockers leads to the reasonable conclusion that otherquorum sensing blockers which are known to exhibit quorum sensingblocking in other systems, such as protease production, will also beeffective against biofilm formation.

[0120] The compounds of the invention are advantageously used to treatand/or prevent infections, such as those caused by V. angufflarum orAeromonas spp. Examples of this type of infection are vibriosis andfurunculosis disease in fish. Inhibition of biofilm formation by thebacteria, optionally together with a reduction or elimination ofextracellular protease production, renders the bacteria substantiallynon-pathogenic. The compounds of the invention may be formulated byconventional methods for use in the treatment and/or prevention ofbacterial infection. For example, the compounds may be used as solid orliquid preparations (such as tablets, suspensions or solutions for oraladministration or sterile injectable compositions), optionally togetherwith pharmaceutically acceptable diluents, carriers or other additives.

[0121] For the treatment of vibriosis or furunculosis disease in fish,the compounds or compositions containing them may be applied directly tothe fish or they may be added to the fish's food or water.

[0122] In another embodiment, the invention provides a method ofremoving a biofilm from a surface which comprises treating the surfacewith a compound of the invention. The surface is preferably the insideof an aqueous liquid distribution system, such as a drinking waterdistribution system or a supply line connected to a dental air-watersystem. The removal of biofilms from this type of surface can beparticularly difficult to achieve. The compound is preferably applied tothe surface as a solution of the compound either alone or together withother materials such as conventional detergents or surfactants.

[0123] A further embodiment of the invention is an antibacterialcomposition comprising a compound of the invention together with abacteriocidal agent. In the antibacterial compositions, the compound ofthe invention helps to remove the biofilm whilst the bacteriocidal agentkills the bacteria. The antibacterial composition is preferably in theform of a solution or suspension for spraying and/or wiping on asurface.

[0124] In yet another aspect, the invention provides an article coatedand/or impregnated with a compound of the invention in order to inhibitand/or prevent biofilm formation thereon. The article is preferably ofplastics material with the compound of the invention distributedthroughout the material.

[0125] III. Description of Nucleic Acids Encoding Proteins Involved inSignaling Factor Biosynthesis

[0126] In accordance with another aspect of the present invention, wehave cloned and characterized the genes responsible for production ofthe signaling molecule of the invention in V. harveyi, S. typhimuriumand E. coli. These genes encode a novel family of proteins responsiblefor autoinducer production. We have designated the members of thisfamily of autoinducer production genes as luxS, specificallyluxS_(E.c.), luxS_(S.t.), and luxS_(V.h.) for E. coli, S. typhimuriumand V. harveyi respectively.

[0127] Mutagenesis of luxS in V. harveyi, S. typhimurium and E. colieliminates production of the signaling molecule in all three species ofbacteria. S. typhimurium could be complemented to full production of themolecule by the introduction of either the E. coli O157:H7 luxS_(E.c.)gene or the V. harveyi BB120 luxS_(V.h.) gene. These results indicatethat both the E. coli and V. harveyi LuxS proteins can function with S.typhimurium cellular components to produce the signaling molecule. E.coli DH5 was only partially complemented to production of the signalingmolecule by the introduction of either the E. coli O157:H7 luxS_(E.c) orthe V. harveyi BB120 luxS_(V.h.) gene. Because in trans expression ofluxS genes in E. coli DH5 did not completely restore signaling moleculeproduction, other biochemical or physiological factors may contribute tosignal production.

[0128] The regulation of signaling molecule production differs betweenpathogenic and non-pathogenic strains. For example E. coli O157:H7strains produce AI-2 at 30° and 37° C. with or without glucose while E.coli K-12 strains do not produce the molecule in the absence of apreferred carbon source. And, all of the E. coli O157 strains testedproduce greater signaling activity than do non-pathogenic E. colistrains. Likewise, pathogenic S. typhimurium 14028 producessignificantly more signaling activity than does S. typhimurium LT2.

[0129] Sequence analysis shows that the LuxS proteins are highlyhomologous, and complementation data suggest that the proteins canfunction across species. These results indicate that the enzymaticactivity carried out by the LuxS proteins and any other cellularmachinery that contributes to synthesis of the signaling molecule areconserved. We did not identify any amino acid sequence motif in the LuxSproteins that is indicative of a particular function. Therefore, theLuxS proteins most likely catalyze one specific enzymatic step inbiosynthesis of the signaling molecule. The remainder of the stepsinvolved in signaling molecule biosynthesis could be a consequence ofnormal intermediary metabolic processes. The luxS genes identified herebear no homology to other genes known to be involved in production ofacyl-homoserine lactone autoinducers (luxI-like (Fuqua et al., J.Bacteriol. 176, 269-275, 1994), luxLM-ainS-like (Bassler et al, 1993,supra; Gilson et al, J. Bacteriol. 177, 6946-6951, 1995), furtherindicating that the signaling molecules of the present invention arenovel.

[0130] Database analysis of finished and unfinished bacterial genomesreveals that many other species of bacteria possess a gene homologous toluxS from V. harveyi, S. typhimurium and E. coli. The species ofbacteria identified and the percent homology/identity (H/I) to the LuxSprotein of V. harveyi are as follows: Haemophilus influenzae (88/72),Helicobacter pylori (62/40), Bacillus subtilis (58/38), Borreliaburgfdorferi (52/32), Neisseria meningitidis (89/80), Neisseriagonorrhoeae (89/80), Yersinia pestis (85/77), Campylobacter jejuni(85/74), Vibrio cholerae (95/90), Deinococcus radiodurans (65/45),Mycobacterium tuberculosis (59/41), Enterococcus faecalis (60/44),Streptococcus pneumoniae (57/36) and Streptococcus pyogenes (57/36). Asreported earlier (Bassler et al., 1997, supra), a few of these specieswere tested for production of the signaling molecule. We showed that V.cholerae and Y. enterocolitica but not B. subtilis produced signalingactivity. We believe that B. subtilis does produce the molecule but theenvironmental conditions that induce its synthesis have not yet beendetermined. Furthermore, we believe that all of the species identifiedin the database analysis produce an AI-2-like molecule.

[0131] The nucleotide sequences of the luxS genes from V. harveyi, E.coli and S. typhimurium are set forth at the end of the specification asSEQ ID NO:1, SEQ ID NO:2 and SEQ ID NOS:3 and 4, respectively (thesequences read in the 5′ to 3′ direction). These genes are sometimesreferred to herein as “LuxS_(V.h.)”, “LuxS_(E.c.)” and “LuxS_(S.t.)”,respectively. The amino acid sequences deduced from SEQ ID NOS: 1-4 areset forth at the end of the specification (and in FIG. 11) as SEQ IDNO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively. It is believed thatSEQ ID NOS:1 and 2 constitute full-length clones, whereas SEQ ID NO:3and SEQ ID NO:4 do not.

[0132] The LuxS genes from V. harveyi, E. coli and S. typhimurium aredescribed in greater detail in Example 3. Although those particular LuxSgenes and their encoded proteins are exemplified herein, this inventionencompasses LuxS genes and their encoded enzymes from any bacterialspecies, having the sequence, structural and functional properties ofthe LuxS-encoded proteins described herein. As mentioned in Example 3,homologous nucleic acid sequences have been identified in a variety ofbacterial species, but identity of those sequences as LuxS genesheretofore had not been appreciated. LuxS nucleotide and deduced aminoacid sequences from other bacterial species are set forth at the end ofthe specification as SEQ ID NOS: 5-9 and 13-17, respectively, andinclude sequences from the following species: Haemophilus influenzae,Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi and Vibriocholerae.

[0133] In addition to LuxS homologs from species other than V. harveyi,E. coli or S. typhimurium, variants and natural mutants of SEQ IDNOS:1-9 are likely to exist within different species or strains ofVibrio, Escherichia and Salmonella (indeed, E. coli strain DH5 possessesa non-functional mutant form of the gene). Because such variants areexpected to possess certain differences in nucleotide and amino acidsequence, this invention provides an isolated LuxS nucleic acid moleculeand encoded protein having at least about 50-60% (preferably 60-80%,most preferably over 80%) sequence homology in the coding region withthe nucleotide sequences set forth as SEQ ID NOS:1-9, respectively (and,preferably, specifically comprising the coding regions of SEQ IDNOS:1-9), and the amino acid sequence of SEQ ID NOS:10-17. Because ofthe natural sequence variation likely to exist among these proteins andnucleic acids encoding them, one skilled in the art would expect to findup to about 40-50% sequence variation, while still maintaining theunique properties of the LuxS-encoded proteins of the present invention.Such an expectation is due in part to the degeneracy of the geneticcode, as well as to the known evolutionary success of conservative aminoacid sequence variations, which do not appreciably alter the nature ofthe protein. Accordingly, such variants are considered substantially thesame as one another and are included within the scope of the presentinvention.

[0134] For purposes of this invention, the term “substantially the same”refers to nucleic acid or amino acid sequences having sequence variationthat do not materially affect the nature of the protein (i.e. thestructural characteristics and/or biological activity of the protein).With particular reference to nucleic acid sequences, the term“substantially the same” is intended to refer to the coding region andto conserved sequences governing expression, and refers primarily todegenerate codons encoding the same amino acid, or alternate codonsencoding conservative substitute amino acids in the encoded polypeptide.With reference to amino acid sequences, the term “substantially thesame” refers generally to conservative substitutions and/or variationsin regions of the polypeptide not involved in determination of structureor function. The terms “percent identity” and “percent similarity” arealso used herein in comparisons among amino acid sequences. These termsare intended to be defined as they are in the UWGCG sequence analysisprogram (Devereaux et al., Nucl. Acids Res. 12: 387-397, 1984),available from the University of Wisconsin, and the parameters used bythat program are the parameters intended to be used herein to comparesequence identity and similarity.

[0135] A. Preparation of LuxS Nucleic Acid Molecules, Encoded Proteins,and Immunologically Specific Antibodies

[0136] 1. Nucleic Acid Molecules

[0137] LuxS Nucleic acid molecules of the invention may be prepared bytwo general methods: (1) They may be synthesized from appropriatenucleotide triphosphates, or (2) they may be isolated from biologicalsources. Both methods utilize protocols well known in the art.

[0138] The availability of nucleotide sequence information, such as theDNAs having SEQ ID NOS:1-9, enables preparation of an isolated nucleicacid molecule of the invention by oligonucleotide synthesis. Syntheticoligonucleotides may be prepared by the phosphoramadite method employedin the Applied Biosystems 38A DNA Synthesizer or similar devices. Theresultant construct may be purified according to methods known in theart, such as high performance liquid chromatography (HPLC). Long,double-stranded polynucleotides, such as a DNA molecule of the presentinvention, must be synthesized in stages, due to the size limitationsinherent in current oligonucleotide synthetic methods. Such longdouble-stranded molecules may be synthesized as several smaller segmentsof appropriate complementarity. Complementary segments thus produced maybe annealed such that each segment possesses appropriate cohesivetermini for attachment of an adjacent segment. Adjacent segments may beligated by annealing cohesive termini in the presence of DNA ligase toconstruct an entire 1.8 kb double-stranded molecule. A synthetic DNAmolecule so constructed may then be cloned and amplified in anappropriate vector.

[0139] LuxS Nucleic acids also may be isolated from appropriatebiological sources using methods known in the art. In a preferredembodiment, a genomic clone is isolated from a cosmid expression libraryof an S. typhimurium or E. coli genome. In another embodiment, a genomicclone is isolated from a cosmid library of another bacterial genome.

[0140] In accordance with the present invention, nucleic acids havingthe appropriate level sequence homology with the protein coding regionof any of SEQ ID NOS:1-9 may be identified by using hybridization andwashing conditions of appropriate stringency. For example,hybridizations may be performed, according to the method of Sambrook etal., using a hybridization solution comprising: 5×SSC, 5×Denhardt'sreagent, 1.0% SDS, 100 g/ml denatured, fragmented salmon sperm DNA,0.05% sodium pyrophosphate and up to 50% formamide. Hybridization iscarried out at 37-42° C. for at least six hours. Followinghybridization, filters are washed as follows: (1) 5 minutes at roomtemperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in 1×SSC and 1% SDS;(4) 2 hours at 42-65° in 1×SSC and 1% SDS, changing the solution every30 minutes.

[0141] One common formula for calculating the stringency conditionsrequired to achieve hybridization between nucleic acid molecules of aspecified sequence homology (Sambrook et al., 1989):

T _(m)=81.5C+16.6Log[Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex

[0142] As an illustration of the above formula, using [N+]=[0.368] and50% formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57C. The T_(m) of a DNA duplex decreases by 1-1.5Cwith every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42C.

[0143] Another way to isolate the luxS nucleic acids is to search thepublicly available databases for the luxS sequence in the bacterialgenome of interest, design PCR primers from the sequence and amplify thegene directly from the chromosome. The PCR product can then be cloned.Alternatively, if the complete sequence of a specific bacterial genomeis not available, the sequences set forth in the present invention, orany other luxS sequence, may be used to design degenerateoligonucleotides for PCR amplification and cloning of luxS from thechromosome.

[0144] Nucleic acids of the present invention may be maintained as DNAin any convenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

[0145] LuxS nucleic acid molecules of the invention include DNA, RNA,and fragments thereof which may be single- or double-stranded. Thus,this invention provides oligonucleotides (sense or antisense strands ofDNA or RNA) having sequences capable of hybridizing with at least onesequence of a nucleic acid molecule of the present invention, such asselected segments of the DNA having SEQ ID NOS:1, 2 or 3. Sucholigonucleotides are useful as probes for detecting LuxS genes ortranscripts.

[0146] 2. Proteins and Antibodies

[0147] A full-length LuxS gene product of the present invention may beprepared in a variety of ways, according to known methods. The proteinmay be purified from appropriate sources, e.g., cultured bacteria suchas S. typhimurium, E. coli or V. harveyi.

[0148] The availability of full-length LuxS nucleic acid moleculesenables production of the encoded protein using in vitro expressionmethods known in the art. According to a preferred embodiment, theenzyme may be produced by expression in a suitable expression system.For example, part or all of a DNA molecule, such as the DNA having SEQID NO:1 or 2, may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli, or a eucaryotic cell,such as Saccharomyces cerevisiae or other yeast. Such vectors comprisethe regulatory elements necessary for expression of the DNA in the hostcell, positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences.

[0149] The protein produced by LuxS gene expression in a recombinantprocaryotic or eucyarotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein. Suchmethods are commonly used by skilled practitioners.

[0150] The protein encoded by the LuxS gene of the invention, preparedby one of the aforementioned methods, may be analyzed according tostandard procedures. For example, the protein may be subjected to aminoacid sequence analysis, according to known methods. The stability andbiological activity of the enzyme may be determined according tostandard methods, such as by the ability of the protein to catalyzeproduction of the signaling molecule under different conditions.

[0151] The present invention also provides antibodies capable ofimmunospecifically binding to the LuxS-encoded protein of the invention.Polyclonal antibodies may be prepared according to standard methods. Ina preferred embodiment, monoclonal antibodies are prepared, which reactimmunospecifically with various epitopes of the protein. Monoclonalantibodies may be prepared according to general methods of Köhler andMilstein, following standard protocols. Polyclonal or monoclonalantibodies that immunospecifically interact with the LuxS-encodedproteins can be utilized for identifying and purifying such proteins.For example, antibodies may be utilized for affinity separation ofproteins with which they immunospecifically interact. Antibodies mayalso be used to immunoprecipitate proteins from a sample containing amixture of proteins and other biological molecules.

[0152] B. Uses of LuxS Nucleic Acid Molecules, Encoded Protein andImmunologically Specific Antibodies

[0153] LuxS nucleic acids may be used for a variety of purposes inaccordance with the present invention. DNA, RNA, or fragments thereofmay be used as probes to detect the presence of and/or expression ofLuxS genes. Methods in which LuxS nucleic acids may be utilized asprobes for such assays include, but are not limited to: (1) in situhybridization; (2) Southern hybridization (3) northern hybridization;and (4) assorted amplification reactions such as polymerase chainreactions (PCR).

[0154] The LuxS nucleic acids of the invention may also be utilized asprobes to identify related genes from other bacteria. As is well knownin the art, hybridization stringencies may be adjusted to allowhybridization of nucleic acid probes with complementary sequences ofvarying degrees of homology.

[0155] As described above, LuxS nucleic acids are also used to advantageto produce large quantities of substantially pure encoded protein, orselected portions thereof. It should be noted in this regard that thecloned genes inserted into expression vectors can be used to make largequantities of the signaling molecule itself, from any selected bacterialspecies, in a recombinant host such as E. coli DH5. Specific luxS genesare cloned, a large quantity of the encoded protein produced, therebyproducing a large quantity of the specific signaling molecule. This willbe particularly useful determining differences in the structures ofsignaling molecules from different species, if such differences arefound to exist. Alternatively, a large quantity of signaling moleculefrom the species of interest could be made using the cloned gene in anexpression vector, and thereafter used in library screens for potentialtargets in petri plate assays, as described above.

[0156] Purified LuxS gene products, or fragments thereof, may be used toproduce polyclonal or monoclonal antibodies which also may serve assensitive detection reagents for the presence and accumulation of thoseproteins in cultured cells. Recombinant techniques enable expression offusion proteins containing part or all of a selected LuxS-encodedprotein. The full length protein or fragments of the protein may be usedto advantage to generate an array of monoclonal or polyclonal antibodiesspecific for various epitopes of the protein, thereby providing evengreater sensitivity for detection of the protein in cells or tissue.

[0157] Other uses of the LuxS proteins include overproduction to make aquantity of the LuxS proteins sufficient for crystallization. Solvingthe crystal structure of the LuxS proteins would enable the exactdetermination of the LuxS active site for catalysis of production of thesignaling molecule. The LuxS crystal structure can therefore be used forcomputer modeling that would greatly facilitate design of signalingmolecule analogs, LuxS inhibitors, and rational drug design in general.

[0158] Polyclonal or monoclonal antibodies immunologically specific fora LuxS-encoded protein may be used in a variety of assays designed todetect and quantitate the protein. Such assays include, but are notlimited to: (1) flow cytometric analysis; (2) immunochemicallocalization of a LuxS protein in cells or tissues; and (3) immunoblotanalysis (e.g., dot blot, Western blot) of extracts from various cellsand tissues. Additionally, as described above, antibodies can be usedfor purification of the proteins (e.g., affinity column purification,immunoprecipitation).

[0159] IV. Vibrio Harveyi Screening Strain

[0160] In another aspect, the invention provides a novel strain ofVibrio Harveyi having a genotype that is luxN⁻, luxS⁻. The The Gramnegative bacterium Vibrio harveyi contains two parallel quorum sensingcircuits which synthesize and detect two different autoinducer molecules(FIG. 13). Circuit 1 synthesizes AI-1 a HSL autoinducer similar instructure to autoinducers synthesized by the LuxI/R pathway found inother gram negative bacteria. Circuit 2 synthesizes AI-2, the structureof which has not been determined. Synthesis of AI-1 and AI-2 isdependent on LuxLM and LuxS respectively. Following the buildup of acritical external concentration of the autoinducers, signaling occursvia a series of a phosphorylation/dephosphorylation reactions. The AI-1and AI-2 detectors, LuxN and LuxQ respectively, contain both a sensorkinase domain with a conserved histidine (H1) and an attached responseregulator domain with a conserved aspartate (D1). Signals from bothsensors are channeled to the shared integrator protein LuxU, which isphosphorylated on a histidine residue (H2). Subsequently, the signal istransduced to a conserved aspartate residue (D2) on the responseregulator protein LuxO. LuxO-phosphate controls the expression of theluciferase structural operon luxCDABE which results in the emission oflight. The presence of either AI-1 or AI-2 is sufficient to turn onlight production in wild-type V. harvyi (strain BB120). For this reason,we have V. harvyi strains containing separate mutations in Lux genes L,M, S or Q which are defective in their ability to synthesize or detectAI-1 or AI-2, respectively. AI-2 is detectable using strain BB170 whichis sensor 1⁻, sensor 2⁺ (LuxN⁻, LuxQ⁺). This strain was used to detectAI-2 in diverse bacteria. The light emission response of wild type,LuxN- and LuxQ-phenotypes to increasing cell density is shown in FIG.14.

[0161] BB170 is a sensitive reporter for AI-2, however, the BB170 strainis not optimal for use as a reporter for inhibitors of the quorumpathway in a microtiter based assay. The desired strain is defective inits ability to detect AI-1 (sensor 1⁻) and defective in its ability tosynthesize AI-2. Thus, the invention provides a strain of V. harveyithat is genotypically luxN⁻ and luxS⁻. The new strain, designated MM32,is useful for identifying inhibitors of the quorum sensing pathway. Forexample, since the new strain is sensor 1⁻, its growth or ability toluminesce will not be affected by those organisms producing AI-1.Further, since MM32 is defective for production of AI-2, the addition ofexogenous AI-2, or analogs thereof, allows for the rapid identificationof inhibitors of AI-2.

[0162] In addition, the materials described above are ideally suited forthe preparation of a kit. Such a kit may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method.

[0163] The container means may comprise a strain of bacteria capable ofdetecting the presence of an autoinducer. Preferably, the bacterialstrain will be capable of providing an easily detectable signal in thepresence of autoinducer-2. More preferably, he desired strain isdefective in its ability to detect AI-1 (sensor 1⁻, and defective in itsability to synthesize AI-2. Thus, the kit may provide a strain of V.harveyi that is genotypically luxN⁻ and luxS⁻ designated MM32. Thebacterial strain is useful for identifying autoinducer-2 as well asinhibitors of autoinducer-2 and the quorum sensing pathway.

[0164] V. Methods for Detecting a Bacterial Biomarker

[0165] Many bacteria presently known to utilize the autoinducer-1signaling factor associate with higher organisms, i.e., plants andanimals, at some point during their lifecycles. For example, Pseudomonasaeruginosa is an opportunistic pathogen in humans with cystic fibrosis.P.aeruginosa regulates various virulence determinants with AI. Otherexamples of AI producing bacteria include Erwinia carotovora,Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi, andAgrobacterium tumefaciens. E. carotovora infects certain plants andcreates enzymes that degrade the plant's cell walls, resulting in whatis called “soft rot disease.” Yersinia enterocolitica is a bacteriumwhich causes gastrointestinal disease in humans and has been reported toproduce an autoinducer. P.aureofaciens associates with the roots ofplants and produces antibiotics that block fungus growth in the roots.The antibiotic synthesis is under autoinducer control. The presentinvention provides novel autoinducer-2 and methods of usingautoinducer-2. In contrast to autoinducer-1, autoinducer-2 is believedto be an intra-species as well as inter-species signaling factor.Autoinducer-2 is further believed to regulate the expression ofpathogenic and virulence factors not regulated by autoinducer-1. Thus,the present invention provides a method to identify and regulate theexpression of bacterial biomarkers in, for example, pathogenic bacteria.Methods of the invention can be used to regulate the activity ofbacterial pathogens that are present in both plants and animals.

[0166] The invention further provides a method for detecting anautoinducer-associated bacterial biomarker by contacting at least onebacterial cell with an autoinducer molecule under conditions and forsuch time as to promote induction of a bacterial biomarker. As usedherein, an “autoinducer-associated bacterial biomarker” is any bacterialcell component which is regulated, modified, enhanced, inhibited orinduced in response to an autoinducer. A biomarker can be any bacterialcell component that is identifiable by known microscopial, histologicalor molecular biological techniques. Such biomarkers can be used, forexample, to distinguish pathogenic from non-pathogenic bacteria. Such abiomarker can be, for example, a molecule present on a cell surface, aprotein, a nucleic acid, a phosphorylation event or any molecular ormorphological characteristic of a bacterial cell that is modified as aresult of the bacterium being contacted with an autoinducer. Preferably,the autoinducer is autoinducer-2. The method of the invention isparticularly useful for identifying a biomarker which is indicative ofbacterial pathogenicity. As previously noted, autoinducers areextracellular signalling factors used by a variety of bacteria toregulate cellular functions in response to various environmentalstimuli, including high population density. It is believed thatpathogenic bacteria express a biomarker, such as an antigenicdeterminant, as a result of increased autoinducer concentration in thesurrounding environment. Thus, the present invention provides a methodfor identifying a biomarker by contacting a bacterium with autoinducer-2and assaying for the presence of the biomarker.

[0167] The method of the invention contemplates the use of a probe toidentify a biomarker present in a bacterial cell. As used herein, a“probe” can be a nucleic acid, protein, small molecule or antibodyuseful for detecting a bacterial biomarker present in a sample. Theprobe can be used in a screening assay to identify a biomarker presentin a sample after the sample has been contacted with, for example, anautoinducer. For example, a bacterial biomarker produced by a bacteriumfollowing contact with an autoinducer can can be identified bycontacting a sample containing the bacterium with a probe that binds tothe biomarker. Such assays can be used to detect, prognose, diagnose, ormonitor various conditions, diseases, and disorders, or monitor thetreatment thereof. A probe can be detectably labeled such that the probeis detectable when bound to its target marker. Such means for detectablylabeling a probe include a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,fluorescent, or radionuclide label. Other reporter means and labels arewell known in the art. In addition, the method of the invention can beused to analyze differential gene expression in a bacterial cellfollowing contact with an autoinducer. For example, where the expressionof genes in different cells, normally a cell of interest and a control,is compared and any discrepancies in expression are identified. In suchassays, the presence of discrepancies indicates a difference in theclasses of genes expressed in the cells being compared. Methods that canbe used to carry out the foregoing are commonly known in the art.

[0168] The present invention provides a method for identifying abiomarker which can be a protein. For example, a bacterial proteinexpressed in response to an autoinducer molecule can be detected usingthe appropriate antibody. The expressed protein can be, for example, anantigenic determinant indicative of a pathogenic bacterium. Antibodiesused in the method of the invention are suited for use, for example, inimmunoassays for the detection of such a determinant. The term“antibody” as used herein is meant to include intact molecules ofpolyclonal or monoclonal antibodies, as well as fragments thereof, suchas Fab and F(ab′)_(2′). For example, monoclonal antibodies are made fromantigen containing fragments of a protein by methods well known to thoseskilled in the art (Kohler, et al., Nature, 256:495, 1975).

[0169] In addition, the monoclonal antibodies in these immunoassays canbe detectably labeled in various ways. For example, radioisotopes may bebound to an immunoglobulin either directly or indirectly by using anintermediate functional group. Intermediate functional groups whichoften are used to bind radioisotopes which exist as metallic ions toimmunoglobulins are the bifunctional chelating agents such asdiethylenetriamine-pentacetic acid (DTPA) and ethylenediaminetetraaceticacid (EDTA) and similar molecules. Typical examples of metallic ionswhich can be bound to monoclonal antibodies are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0170] A probe useful in the method of the invention can also be anucleic acid probe. For example, nucleic acid hybridization techniquesare well known in the art and can be used to identify an RNA or DNAbiomarker present in a sample containing a bacterium contacted with anautoinducer. Screening procedures which rely on nucleic acidhybridization make it possible to identify a biomarker from any sample,provided the appropriate probe is available. For example,oligonucleotide probes, which can correspond to a part of the sequenceencoding a target protein, can be synthesized chemically. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. For suchscreening, hybridization is preferably performed under in vitro or in invivo conditions known to those skilled in the art.

[0171] In addition, the materials described above are ideally suited forthe preparation of a kit. Such a kit may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. A kitof the invention may contain a first container means comprising isolatedautoinducer-2. The isolated autoinducer-2 can be used to regulate theexpression of a biomarker in a target bacterium. For example,autoinducer-2 can be used to induce expression of a particular biomarkerwhich can then be identified by a probe. Thus, the kit may contain asecond container means comprising a probe that can be detectablylabeled. The kit may also have a third container comprising areporter-means, such as a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,fluorescent, or radionuclide label. Other reporter means and labels arewell known in the art. For example, the kit of the invention may providereagents necessary to perform nucleic acid hybridization analysis asdescribed herein or reagents necessary to detect antibody binding to atarget.

[0172] The following description sets forth the general proceduresinvolved in practicing this aspect of the present invention. To theextent that specific materials are mentioned, it is merely for purposesof illustration and is not intended to limit the invention. Unlessotherwise specified, general cloning procedures, such as those set forthin Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory(1989) (hereinafter “Sambrook et al.”) or Ausubel et al. (eds) CurrentProtocols in Molecular Biology, John Wiley & Sons (1998) (hereinafter“Ausubel et al.”) are used.

EXAMPLE 1 Quorum Sensing in Escherichia coli and Salmonella typhimurium

[0173] There have been preliminary indications that E. coli senses celldensity (Huisman et al., Science 265: 537-539, 1994; Sitnikov et al.,Proc. Natl. Acad. Sci. USA 93: 336-341, 1996; Garcia-Lara et al., J.Bacteriol. 178: 2742-2748, 1996). We took advantage of the reducedselectivity of the Signaling System 2 sensor in V. harveyi to develop asensitive assay for detection of extracellular signal molecules producedby E. coli and S. typhimurium. Using this assay we could determine theconditions under which many strains of E. coli and S. typhimuriumsynthesize, secrete, and degrade a signaling substance that willinteract with the V. harveyi System 2 detector.

[0174] Materials and Methods

[0175] Preparation of cell-free culture fluids. E. coli strains AB1157and DH5 and S. typhimurium strain LT2 were grown at 30° C. overnightwith aeration in LB broth containing glucose at the concentrationsspecified in the text. The following morning fresh LB medium containingthe same concentration of glucose used for the overnight growth wasinoculated at a 1:100 dilution with the overnight grown cultures. Thefresh cultures were grown for various times at 30° C. with aeration.Cell-free culture fluids were prepared by removing the cells from thegrowth medium by centrifugation at 15,000 rpm for 5 min in amicrocentrifuge. The cleared culture fluids were passed through 0.2 m HTTuffryn filters (Gelman) and stored at −20° C. Cell-free culture fluidscontaining V. harveyi Autoinducer-2 were prepared from V. harveyi strainBB152 (Autoinducer 1⁻, Autoinducer 2⁺). V. harveyi BB120 (Autoinducer1⁺, Autoinducer 2⁺) was used to prepare culture fluids containingAutoinducer-1. In both cases, the V. harveyi strains were grownovernight at 30° C. with aeration in AB (Autoinducer Bioassay) (Bassleret al., 1993, supra) medium. Cell-free culture fluids from V. harveyiwere prepared from the overnight culture exactly as described above forE. coli and S. typhimurium.

[0176] Assay for Production Of Signaling Molecules.

[0177] Cell-free culture fluids from E. coli, S. typhimurium and V.harveyi strains were tested for the presence of signaling substancesthat could induce luminescence in the V. harveyi reporter strain BB170or BB886. In the assays, 10 l of cell-free culture fluids from E. coliAB1157, E. coli DH5, and S. typhimurium LT2 strains grown and harvestedas described above were added to 96-well microtiter dishes. The V.harveyi reporter strain BB170 or BB886 was grown for 16 h at 30° C. withaeration in AB medium, diluted 1:5000 into fresh AB medium, and 90 l ofthe diluted cells were added to the wells containing the E. coli and S.typhimurium cell-free culture fluids. Positive control wells contained10 l of cell-free culture fluid from strain V. harveyi BB152(Autoinducer-1⁻, Autoinducer-2⁺) or V. harveyi BB120 (Autoinducer-1⁺,Autoinducer-2⁺). Negative control wells contained 10 l of sterile growthmedium. The microtiter dishes were shaken in a rotary shaker at 175 rpmat 30° C. Every hour, light production was measured using a Wallac Model1450 Microbeta Plus liquid scintillation counter in thechemiluminescence mode. The V. harveyi cell density was measured bydiluting the same aliquots of cells used for measuring luminescence,spreading the dilutions onto solid LM medium (Bassler et al., 1993,supra), incubating the plates overnight at 30C, and counting theresulting colonies the following day.

[0178] Preparation of E. coli and S. typhimurium viable and UV-killedcells for the activity assay. E. coli AB1157, E. coli DH5 and S.typhimurium LT2 cultures were grown for 8 h in LB containing 0.5%glucose at 30° C. with aeration. The cultures were subjected tocentrifugation for 5 min at 15,000 rpm in a microcentrifuge and thegrowth medium was removed from the cell pellets by aspiration. The cellpellets were resuspended in AB medium and washed by vigorous mixing. Thecells were again subjected to centrifugation for 5 min at 15,000 rpm.The AB wash medium was removed and discarded and the cells wereresuspended in fresh AB medium. Each cell suspension was diluted to give1×10⁶ cells/10 l, and multiple 10 l aliquots were added to wells ofmicrotiter dishes. Half of the cell aliquots were treated with shortwavelength ultraviolet light for 15 min at a distance of 10 cm. Thistreatment was sufficient to kill all of the cells as judged by platingand incubating the UV-treated cells, and ensuring that no growthoccurred by the next day. 90 l of the diluted V. harveyi reporter strainBB170 was next added to the wells containing either the viable or deadE. coli and S. typhimurium cells, and the activity assay was carried outexactly as described in the previous section.

[0179] Analysis of glucose in S. typhimurium LT2 culture fluids. Glucoseconcentrations were determined in cell-free culture fluids prepared fromS. typhimurium using a Trinder assay (Diagnostic Chemicals Ltd.)according to the recommendations of the manufacturer, except that theglucose standards were prepared in LB medium. The assay was sensitive toless than 0.002% glucose. No interfering substances were present in LBmedium or spent LB culture fluids.

[0180] Results and Discussion

[0181]E. coli AB1157 and S. typhimurium LT2 produce a signalingsubstance that specifically induces one of the two quorum sensingsystems of V. harveyi. The V. harveyi reporter strain BB170 has thequorum sensing phenotype Sensor 1⁻, Sensor 2⁺. It induces lux expressionin response to extracellular signals that act exclusively through theSignaling System 2 detector. Addition of 10% cell-free spent culturefluid prepared from V. harveyi strain BB152 (which contains the System 2autoinducer) stimulates the reporter strain roughly 1000-fold over theendogenous level of luminescence expression. In FIG. 1, the lightproduction by V. harveyi BB170 induced by the addition of 10% cell-freespent culture fluids is normalized to 100% activity.

[0182]E. coli strain AB1157 and S. typhimurium strain LT2 were grown for8 h in LB broth or LB broth containing 0.5% glucose. The E. coli and S.typhimurium cells were removed from the growth medium and the cell-freeculture fluids were prepared and assayed for an activity that couldinduce luminescence expression in V. harveyi. Addition of 10% cell-freeculture fluid from S. typhimurium LT2 or E. coli AB1157 grown in LBcontaining glucose maximally induced luminescence in the reporter strainBB170, similar to culture fluids from V. harveyi BB152 (FIG. 1A).Specifically, E. coli AB1157 produced 106% and S. typhimurium produced237% of the V. harveyi BB1S2 activity. When the E. coli and S.typhimurium were grown in LB without added glucose they did not producethe signaling factor. Substitution of 10% (v/v) of LB medium containing0.5% glucose did not stimulate luminescence in the reporter strain,indicating that there is no substance in the LB-glucose growth mediumthat induces luminescence expression in V. harveyi. We tested obviouscandidates for the signal including glucose, amino acids, cAMP, acetate,homoserine lactone, -ketoglutarate and other keto acids that are knownto be excreted. None of these compounds has activity. These resultssuggest that V. harveyi BB170 can respond to some substance secreted byE. coli AB1157 and S. typhimurium LT2 when they are grown on LBcontaining glucose.

[0183] Analogous experiments were performed with the V. harveyi reporterstrain BB886 (Sensor 1⁺, Sensor 2⁻). V. harveyi BB886 is defective inits response to signaling molecules that act through the SignalingSystem 2 detector, but it is an otherwise wild type strain (Bassler etal., Mol. Microbiol. 13: 273-286, 1994). FIG. 1B shows the normalized100% activation of V. harveyi BB886 by cell-free spent culture fluidsprepared from V. harveyi BB120. V. harveyi BB120 produces the System 1autoinducer N-(3-hydroxybutanoyl)-L-homoserine lactone (Bassler et al.,1993, supra). Addition of S. typhimurium LT2 and E. coli AB1157cell-free culture fluids to V. harveyi strain BB886 caused a 5% and a 1%increase above the control level (FIG. 1B). Together the results ofFIGS. 1A and 1B show that the signaling molecule produced by E. coli andS. typhimurium must act specifically through V. harveyi Signaling System2 and not some other, unidentified pathway.

[0184] Viable E. coli AB1157 and S. typhimurium LT2 are required forsecretion of the signaling molecule. We considered the possibility thatgrowth of E. coli AB1157 and S. typhimurium LT2 in LB medium containingglucose simply allowed them to utilize and therefore remove somepre-existing inhibitor of induction of luminescence. To show that thecells themselves produce the soluble signaling factor, we added washedE. coli and S. typhimurium cells directly to the luminescence assay.These results are presented in FIG. 2. In this experiment, E. coliAB1157 and S. typhimurium LT2 were grown for 8 h in LB containing 0.5%glucose; the conditions for maximal production of the signaling factor.The cells were removed from the LB-glucose growth medium bycentrifugation, and sterile V. harveyi luminescence assay medium wasused to wash and resuspend the cell pellets. 1×10⁶ E. coli AB1157 or S.typhimurium LT2 cells were added to the diluted V. harveyi BB170 cultureat the start of the experiment. In FIG. 2, the left-hand bar in eachseries shows that the presence of washed E. coli AB1157 or S.typhimurium LT2 cells is sufficient to fully induce luminescence in V.harveyi BB170. E. coli AB1157 and S. typhimurium LT2 stimulated luxexpression in V. harveyi BB170 821-fold and 766-fold respectively.Identical aliquots of the washed E. coli or S. typhimurium cells werekilled with short wave ultraviolet light prior to addition to the assay.When dead cells were included in the assay, no stimulation ofluminescence occurred. In FIG. 2, these results are shown in theright-hand bar for each strain. Taken together, the results show thatthe stimulatory factor is produced by the E. coli AB1157 and S.typhimurium LT2 cells themselves during the time course of theexperiment; the factor could not have come from the medium in which thecells had been grown. This factor is actively released into the mediumby E. coli and S. typhimurium because dead cells have no activity.

[0185]E. coli DH5 does not produce the signaling activity. Clinicalisolates of E. coli and Salmonella also produce the signaling compound.Ten clinical isolates of Salmonella and five pathogenic isolates of E.coli O157 were assayed and all produced the activity. It was conceivablethat the signal was some normal byproduct of glucose metabolism thatsimply diffuses out of the cells. This is not the case however, becausewe show that E. coli DH5, which is equally capable of utilizing glucoseas E. coli AB1157 and S. typhimurium LT2, does not produce the signalingactivity. FIG. 1A demonstrates that unlike E. coli AB1157 and S.typhimurium LT2, the addition of 10% cell-free culture fluid preparedfrom E. coli DH5 grown 8 h in LB containing 0.5% glucose does notstimulate light production in V. harveyi BB170. Similarly, inclusion ofwashed viable or killed E. coli DH5 cells in the luminescence assay doesnot stimulate V. harveyi BB170 to produce light (FIG. 2). The inabilityof E. coli DH5 to produce the activity indicates that this highlydomesticated strain lacks the gene or genes necessary for either theproduction or the export of the signaling activity. We assayed otherlaboratory strains of E. coli for the signaling activity (Table 1). OnlyE. coli DH5 was completely defective in producing the extracellularsignal. TABLE 1 Species and Strain Induction of luminescence (%) V.harveyi V. harveyi BB152 100 Salmonella S. typhimurium LT2 237 E. coliE. coli AB1157 106 E. coli DH5 5 E. coli JM109 76 E. coli MG1655 100 E.coli MC4100 93 # by cell-free culture fluids from V. harveyi, S.typhimurium and E. coli is # shown. Cell-free culture fluids wereprepared from various strains of # V. harveyi, S. typhimurium and E.coli as described and tested for # production of a signaling substancethat could stimulate light production in # the reporter strain V.harveyi BB170. The level of V. harveyi stimulation was # normalized to100%. The data for the 5 h time point are shown.

[0186] Glucose regulates the production and degradation of the signalingfactor by S. typhimurium LT2. Cell-free culture fluids from S.typhimurium LT2 and E. coli AB1157 cells grown in LB without addedglucose did not stimulate the expression of luminescence in the reporterstrain, indicating that metabolism of glucose is necessary for theproduction of the signal. We tested other carbohydrates, and in general,growth in the presence of PTS sugars (see Postma et al., in Escherichiacoli and Salmonella Cellular and Molecular Biology, (F. C. Niehardt,ed), Am. Soc. Microbiol., Washington D.C., pp. 1149-1174, 1996) enabledE. coli AB1157 and S. typhimurium LT2 to produce the signal. Of thesugars tested, growth on glucose induced the synthesis of the highestlevel of activity. Growth on other carbon sources, for example TCA cycleintermediates and glycerol, did not induce significant production of thesignaling activity.

[0187] We tested whether the presence of glucose was required for thecells to continue to produce the signal. FIG. 3 shows results with S.typhimurium LT2 grown in LB containing limiting (0.1%) and non-limiting(0.5%) glucose concentrations. FIG. 3A shows that when glucose islimiting, S. typhimurium LT2 produces the signal in mid-exponentialphase (after 4 h growth), but stops producing the signaling activityonce glucose is depleted from the medium. FIG. 3B shows that whenglucose does not become limiting, S. typhimurium LT2 produces greatertotal activity and continues to produce the signaling activitythroughout exponential phase, with maximal activity at 6 h growth.Furthermore, the Figure also shows that the signaling activitysynthesized by mid-exponential phase cells is degraded by the time thecells reach stationary phase. In conditions of limiting glucose, noactivity remained at stationary phase, and when glucose was plentiful,only 24% of the activity remained. Increasing the concentration ofglucose in the growth medium did not change these results, i.e., theactivity was secreted during mid-exponential growth and severely reducedactivity remained in the spent culture fluids by stationary phase.

[0188] In sum, the results presented in this example show that E. coliand S. typhimurium produce a signaling substance that stimulates onespecific quorum sensing system in V. harveyi. Many other bacteria havepreviously been assayed for such an activity, and only rarely werespecies identified that are positive for production of this factor(Bassler et al., 1997, supra). Furthermore, as shown here, the E. coliand S. typhimurium signal is potent, these bacteria make activity equalto that of V. harveyi. The degradation of the E. coli and S. typhimuriumsignal prior to stationary phase indicates that quorum sensing in thesebacteria is tuned to low cell densities, suggesting that quorum sensingin E. coli and S. typhimurium is modulated so that the response to thesignal does not persist into stationary phase. Additionally, quorumsensing in E. coli and S. typhimurium is influenced by severalenvironmental factors. The production and the degradation of the signalare sensitive not only to growth phase but also to the metabolicactivity of the cells. These results indicate that the quorum sensingsignal in E. coli and S. typhimurium has two functions; it allows thecells to communicate to one another their growth phase and also themetabolic potential of the environment.

[0189] Understanding the regulation of quorum sensing in E. coli and S.typhimurium is important for understanding community structure andcell-cell interactions in pathogenesis. In the wild, pathogenic E. coliand S. typhimurium may never reach stationary phase because dispersionis critical. It is therefore appropriate that quorum sensing in E. coliand S. typhimurium should be functioning at low cell density. Thissituation is in contrast to that of V. fischeri, the luminescent marinesymbiont, where the quorum sensing system is only operational at highcell densities; cell densities indicative of existence inside thespecialized light organ of the host. The specific quorum sensing systemsof V. fischeri and E. coli and S. typhimurium appear appropriatelyregulated for the niche in which each organism exists. In both cases,quorum sensing could be useful for communicating that the bacteriareside in the host, not free-living in the environment. Additionalcomplexity exists in the E. coli and S. typhimurium systems becausethese bacteria channel both cell density information and metabolic cuesinto the quorum sensing circuit. Again, signals relaying informationregarding the abundance of glucose or other metabolites couldcommunicate to the bacteria that they should undergo the transition froma free-living mode to the mode of existence inside the host.

[0190] Under all the conditions we have tested, the signaling activitydescribed in this example does not extract quantitatively into organicsolvents and it does not bind to either a cation or anion exchangecolumn. Preliminary characterization indicates that the signal is asmall (less than 1000 MW) polar but apparently uncharged organiccompound. The activity is acid stabile and base labile, it is heatresistant to 80 but not 100° C. Purification of the E. coli, S.typhimurium and V. harveyi signal is described in greater detail in thefollowing examples.

EXAMPLE 2 Regulation of Autoinducer Production in Salmonella typhimurium

[0191] In this example, the conditions under which S. typhimurium LT2produces AI-2, the extracellular factor that stimulates lux expressionin the V. harveyi Sensor 1⁻, Sensor 2⁺ reporter strain, are elucidated.Production of the signaling molecule by S. typhimurium occurs duringgrowth on preferred carbohydrates that, upon utilization by thebacteria, result in a decrease in the pH of the medium. Lowering the pHof the growth medium in the absence of a preferred carbon source induceslimited production of the factor, indicating that the cells areinfluenced by both the changing pH and the utilization of the carbonsource. The signaling activity is degraded by the time the cells reachstationary phase, and protein synthesis is required for degradation ofthe activity. Osmotic shock following growth on an appropriate carbonsource greatly increases the amount of activity present in the S.typhimurium culture fluids. This increased activity is apparently due toinduction of synthesis of the autoinducer and repression of degradationof the activity. E. coli and S. typhimurium possess a protein calledSdiA which is homologous to LuxR from V. fischeri (Wang et al., EMBO J.10: 3363-3372, 1991; Ahmer et al., J. Bacteriol. 180: 1185-1193, 1998).SdiA is proposed to respond to an extracellular factor (Sitnikov et al.,Proc. Natl. Acad. Sci. USA 93: 336-341, 1996; Garcia-Lara et al., J.Bacteriol. 178: 2742-2748, 1996), and it has been shown to controlvirulence factor production in S. typhimurium (Ahmer et al., 1998,supra). The analysis set forth below shows that the AI-2 autoinducersignaling activity does not function through the SdiA pathway.

[0192] Materials and Methods

[0193] Strains and Media. The bacterial strains used in this study andtheir genotypes and phenotypes are listed in Table 2. TABLE 2 Bacterialstrains; their genotypes and relevant phenotypes. Strain GenotypeRelevant Phenotype S. typhimurium Wild LT2 type E. coli 0157 Wild typeE. coli MG1655 F⁻, ilvG, rfb-50 Wild type E. coli MC4100 (lac)U169,araD139, rpsL, LacZ⁻ thi E. coli DH5 supE44, hsdR17, recA1, AI-2⁻ endA1,gyrA96, thi-1, relA1 V. harveyi BB170 luxN::Tn5 Sensor 1⁻, Sensor 2⁺ V.harveyi BB152 luxL::Tn5 AI-1⁻, AI-2⁺ V. harveyi luxN::Cm^(r) Sensor 1⁻,Sensor 2⁺ JAF305

[0194] Luria broth (LB) contained 10 g Bacto Tryptone (Difco), 5 g YeastExtract (Difco) and 10 g NaCl per liter (Sambrook et al., 1989). Therecipe for Autoinducer Bioassay (AB) medium has been reported previously(Greenberg et al., Arch. Microbiol. 120: 87-91, 1979). LM medium(L-Marine) contains 20 g NaCl, 10 g Bacto Tryptone, 5 g Bacto YeastExtract and 15 g Agar per liter (Bassler et al., 1994, supra).Regulation of AI-2 production similar to that reported here was alsoobserved with the ATCC strain Salmonella enterica Serovar Typhimurium14028, an independent clinical isolate of Salmonella enterica SerovarTyphimurium, and nine other Salmonella enterica serovars (other thanTyphimurium).

[0195] Growth conditions for S. typhimurium LT2 and preparation ofcell-free culture fluids. S. typhimurium LT2 was grown overnight in LBbroth with shaking at 30° C. The next day, 30 l of the overnight culturewas used to inoculate 3 ml of fresh LB broth. In cultures containingadditional carbon sources, at the time of inoculation, 20% sterile stocksolutions were added to give the specified final concentrations.Following subculturing of the cells, the tubes were shaken at 200 rpm at30C for the time periods indicated in the text. Cell-free culture fluidswere prepared by removal of the cells from the culture medium bycentrifugation for 5 min at 15,000 rpm in a microcentrifuge. The clearedsupernatants were passed through 0.2 m cellulose acetate Spin X filters(CoStar, Cambridge, Mass.) by centrifugation for 1 min at 8000×g.Samples were stored at −20C. Similar results to those reported here wereobtained when we grew the S. typhimurium at 37° C. The preparation ofcell-free culture fluids from V. harveyi strains has already beenreported (Bassler et al., 1993, supra; Bassler et al., 1997, supra).

[0196] Density-dependent bioluminescence assay. The V. harveyi reporterstrain BB170 (Sensor 1⁻, Sensor 2⁺) (Bassler et al., 1993, supra) wasgrown for 12 h at 30° C. in AB medium, and diluted 1:5000 into fresh ABmedium. Luminescence was measured as a function of cell density byquantitating light production at different times during growth with aWallac Model 1409 liquid scintillation counter (Wallac Inc.,Gaithersburg, Md.). The cell density was measured by diluting the samealiquots of cells used for measuring luminescence, spreading thedilutions onto solid LM medium, incubating the plates overnight at 30°C., and counting the resulting colonies the following day. RelativeLight Units are (counts min⁻¹ ml⁻¹×10³)/(colony forming units ml⁻¹).Cell-free culture supernatants from V. harveyi or S. typhimurium strainswere added to a final concentration of 10% (v/v) at the time of thefirst measurement. In control experiments, 10% (v/v) of AB medium, LBmedium or LB medium containing 0.5% glucose was added instead ofcell-free culture fluids.

[0197]S. typhimurium autoinducer activity assay. The quorum sensingsignaling activity released by S. typhimurium LT2 was assayed followinggrowth under various conditions. 10 l of cell-free culture fluids fromS. typhimurium LT2 grown and harvested as described above were added to96-well microtiter dishes. The V. harveyi reporter strain BB170 wasgrown overnight and diluted as described above. 90 l of the diluted V.harveyi cells were added to the wells containing the S. typhimuriumcell-free culture fluids. Positive control wells contained 10 l ofcell-free culture fluid from V. harveyi BB152 (A1-1⁻, AI-2⁺) (Bassler etal., 1993, supra). The microtiter dishes were shaken in a rotary shakerat 200 rpm at 30° C. Light production was measured hourly using a WallacModel 1450 Microbeta Plus liquid scintillation counter designed formicrotiter dishes (Wallac Inc., Gaithersburg, Md.). In theseexperiments, the cell density was not measured at each time point.Rather, to ensure that increased light production was due to a signalingactivity and not a growth medium component, the luminescence productionby V. harveyi in wells containing cell-free culture fluids was comparedto that produced by V. harveyi in wells containing 10 l of the identicalgrowth medium alone. Data are reported as fold-stimulation over thatobtained for growth medium alone.

[0198] Factors controlling signal production in S. typhimurium. S.typhimurium LT2 was grown for 6 h in LB containing 0.5% glucose asdescribed above. The mid-exponential phase culture was divided intoseveral identical aliquots. One aliquot of cells was grown to stationaryphase (24 h at 30° C. with shaking). In the remaining aliquots, thecells were removed from the LB-glucose growth medium by centrifugationfor 5 min at 15,000 rpm in a microcentrifuge. The resulting cell pelletswere resuspended at an OD₆₀₀ of 2.0 in either LB, LB+0.5% glucose, LB atpH 5.0, or in 0.1M NaCl, or 0.4M NaCl (in water). The resuspended cellswere shaken at 30° C. or 43° C. for 2 h. Cell-free fluids were preparedfrom the stationary phase culture, and from the cells that had beenresuspended and incubated in the various media or the osmotic shocksolutions. The cell-free fluids were tested for signaling activity inthe S. typhimurium activity assay as described above.

[0199] Effects of growth phase, pH, glucose concentration and osmolarityon autoinducer production by S. typhimurium. S. typhimurium LT2 wasgrown at 30° C. for various times in LB containing limiting (0.1%) andnon-limiting (1.0%) glucose concentrations. At the times specified inthe text, the cell number was determined by plating dilutions of the S.typhimurium cultures onto LB medium and counting colonies the followingday. The pH of the two cultures was measured, and the percent glucoseremaining in each culture was determined using the Trinder assay asdescribed in Example 1. Cell-free culture fluids were prepared from theLB-glucose cultures as described above. The same cells from which thecell-free culture fluids were prepared were resuspended in 0.4M NaClosmotic shock solution and shaken at 200 rpm, 30° C. for 2 h. Wedetermined that this timing was optimal for production of autoinducer.The cells were removed from the osmotic shock solution by centrifugationat 15,000 rpm for 5 min in a microcentrifuge. Cell-free osmotic shockfluids were prepared from the resuspended cells exactly as described forcell-free culture fluids. Signaling activity in both the cell-freeculture fluids and the cell-free osmotic shock fluids was assayed asdescribed above. In experiments in which the pH was maintained at 7.2,the cells were grown in LB+0.5% glucose containing 50 mM MOPS at pH 7.2.The pH was adjusted every 15-30 min using 1 M MOPS pH 7.2. Inexperiments performed at pH 5.0, LB broth was maintained between pH 5.0and 5.2 with 1M NaOH.

[0200] Requirement for protein synthesis in signal production, releaseand degradation by S. typhimurium LT2. S. typhimurium LT2 was pre-grownin LB containing 0.5% glucose at 30° C. to an OD₆₀₀ of 2.5(approximately 6-8 h) The culture was divided into four identicalaliquots. Two aliquots were treated with 100 g/ml Cm for 5 min at roomtemperature after which the cells were harvested by centrifugation at15,000 rpm for 5 min. One Cm-treated cell pellet was resuspended in 0.1MNaCl containing 30 g/ml Cm, and the second pellet was resuspended in0.4M NaCl containing 30 g/ml Cm. Each of these pellets was resuspendedto a final OD₆₀₀ of 2.0. The remaining two culture aliquots were nottreated with Cm. Instead, the cells in these two aliquots were harvestedby centrifugation and resuspended in 0.1M and 0.4M NaCl exactly asdescribed for the Cm-treated cells. The cell suspensions were incubatedat 30° C. with shaking. At the times indicated in the text, 1.5 mlaliquots were removed from the cell suspensions and cell-free osmoticshock fluids were prepared by the procedure described above.

[0201] Analysis of the effect of autoinducer on SdiA regulated geneexpression. A sequence that includes the ftsQ1p and ftsQ2p promoters(Wang et al., 1991, supra) was amplified from E. coli MG1655 chromosomalDNA using the following primers: ftsQ1p,5′-CGGAGATCTGCGCTTTCAATGGATAAACTACG-3′; ftsQ2p,5′-CGCGGATCCTCTTCTTCGCTGTTTCGCGTG-3′. The amplified product containedboth the ftsQ promoters and the first 14 codons of the ftsQ gene flankedby BamHI and BglII sites. The ftsQ1p2p PCR product was cloned into theBamHI site of vector pMLB1034 (Silhavy et al., Experiments with GeneFusions, Cold Spring Harbor Press, 1984) to generate a lacZ fusion thatcontained the promoters, ribosome binding site, and initiation codon offtsQ. A correctly oriented clone, pMS207, and a clone containing theftsQ1p2p insert in the opposite orientation, pMS209, were chosen forfurther analysis. Both inserts were sequenced to ensure that no errorswere introduced during the PCR reaction.

[0202] For ftsQ regulation in E. coli, the plasmids pMS207 and pMS209were transformed into E. coli strain MC4100 (Silhavy et al., 1984,supra), and the transformants were grown overnight in LB containing 100mg/L ampicillin at 30° C. with aeration. For rck regulation, S.typhimurium strains BA1105 (rck::MudJ) and BA1305 (rck::MudJ sdiA) weregrown overnight in LB containing 100 mg/L kanamycin at 30° C. withaeration. The overnight cultures were diluted 20-fold into fresh mediumand grown for an additional 4.5 h. At this time, each culture wasdivided into five identical aliquots and 10% (v/v) of one of thefollowing was added to each aliquot: LB, 0.4M NaCl, 0.4M osmotic shockfluids from S. typhimurium LT2, E. coli O157 or E. coli strain DH5(negative control). The osmotic shock fluids were prepared as describedabove, following pre-growth of the S. typhimurium LT2 and E. coli in LBcontaining 0.5% glucose for 6 h. The cell suspensions were incubated at30° C. for 2 h, after which standard -galactosidase reactions wereperformed on the samples (Miller, A Short Course in Bacterial Genetics,Cold Spring Harbor Laboratory Press, 1992).

[0203] Results

[0204]S. typhimurium LT2 produces an autoinducer-like activity. InExample 1 it was demonstrated that S. typhimurium and E. coli strainsproduce a signaling activity that stimulates lux expression in V.harveyi, and the signaling molecule acts exclusively through the V.harveyi quorum sensing System 2. FIG. 4 shows the induction ofluminescence in the V. harveyi System 2 reporter strain BB170 (Sensor1⁻, Sensor 2⁺). The characteristic quorum sensing behavior of V. harveyiis shown in the control experiment (closed circles). Immediately afterdilution into fresh medium, the light emitted per cell by V. harveyidrops rapidly, over 1000-fold. At a critical cell density, whichcorresponds to the accumulation of a critical concentration ofendogenously produced autoinducer (AI-2) in the medium, the luminescenceper cell increases exponentially, approximately 3 orders of magnitude,to again reach the pre-dilution level.

[0205] Addition of 10% cell-free culture fluid prepared from V. harveyiBB152 (AI-1⁻, AI-2⁺) caused the reporter strain to maintain a high levelof light output following dilution (open circles). The increased lightoutput is due to the V. harveyi BB170 cells responding to the presenceof AI-2 in the cell-free culture fluids prepared from V. harveyi strainBB152 (Bassler et al., 1993, supra). Similarly, addition of cell-freeculture fluid from S. typhimurium LT2 grown in LB+0.5% glucose inducedluminescence in the reporter strain approximately 800-fold over thecontrol level (solid squares). No activity similar to V. harveyi AI-1was produced by S. typhimurium LT2 under these conditions and there isno AI-1 or AI-2 activity in LB+0.5% glucose (see Example 1).

[0206] Environmental factors influence autoinducer production anddegradation in S. typhimurium. Control of autoinducer production in S.typhimurium is different than in other described quorum sensing systems.FIG. 5A demonstrates three important aspects of the regulation ofautoinducer production in S. typhimurium. First, no autoinducer activityis observed when S. typhimurium is grown for 6 h in LB in the absence ofglucose. Second, growth in the presence of glucose for 6 h results insubstantial production of autoinducer (760-fold activation of thereporter strain). Third, activity, while detectable, is severely reducedwhen the S. typhimurium culture is allowed to grow to stationary phase(33-fold activation of the reporter strain).

[0207] We subjected S. typhimurium LT2 to several different treatmentsincluding some environmental stresses in order to begin to understandwhat conditions favor autoinducer production versus those that favorautoinducer degradation. In the experiment presented in FIG. 5B, the S.typhimurium cells were induced for signal production by pre-growth in LBcontaining 0.5% glucose for 6 h. We have shown that under theseconditions, the glucose is not depleted (Surette and Bassler, 1998).After the induction phase of growth, the culture fluid was removed andaliquots of the cells were resuspended and incubated for 2 h under avariety of conditions that are described in the description of FIG. 2.Following each of these treatments cell-free fluids were prepared andtested for activity on BB170.

[0208] It is important to note that in the results presented in FIG. 5B,the S. typhimurium were pre-induced for autoinducer production at thestart of the experiment, i.e., their cell-free culture fluid activatedthe reporter strain 760-fold. FIG. 5B shows that removal of thepre-growth culture fluid from these cells and resuspension of the cellsin LB without glucose, in 0.1 M NaCl (hypotonic conditions), or heatshock at 43° C. for 2 h resulted in no or very low autoinducerproduction. These results indicate that the above treatments result intermination of autoinducer production, or degradation of newly releasedautoinducer, or both.

[0209] In contrast to the above results, resuspension of pre-inducedcells in fresh LB+glucose resulted in continued high-level production ofautoinducer (735-fold activation of the reporter). Similarly, acidic pHpromoted continued production of autoinducer (600-fold activation), andhypertonic osmotic shock (0.4M NaCl) resulted in 1300-fold induction ofthe reporter. Increased AI-2 activity was only observed in the pH 5.0fluids or 0.4M NaCl osmotic shock fluids of cells that were alreadyactively producing AI-2, i.e., if glucose was not included during thepre-growth, no measurable activity was produced following the identical2 h treatments.

[0210] Shifting S. typhimurium cells from LB+glucose to 0.4M NaClresulted in an accumulation of AI-2 activity to a level much greaterthan that observed under any other condition tested. Below it is shownthat S. typhimurium cells resuspended in 0.4M NaCl increase thebiosynthesis and/or release of autoinducer, and furthermore theyapparently do not degrade significant quantities of the releasedactivity. A similar increase in AI-2 production occurs when the S.typhimurium cells are resuspended in 0.4M NaCl, 0.4M KCl or 0.8Msucrose, indicating that the NaCl effect on AI-2 production is anosmotic one, not an ionic one. This apparent osmotic shock effect on theS. typhimurium cells was extremely useful because it enabled us tomeasure maximal release of autoinducer activity in the absence of lossdue to degradation.

[0211] The effect of glucose on signal production in S. typhimurium. InExample 1 we showed that the continued presence of glucose was requiredfor S. typhimurium to produce the quorum sensing signaling factor.Because sugar utilization both increases the growth rate whiledecreasing the pH of the culture, we further analyzed the effect ofmetabolism of glucose, decreasing pH and increasing cell number onsignal production by S. typhimurium. In the experiment presented in FIG.6, we measured signal production, growth rate, and pH in growing S.typhimurium LT2 cultures containing limiting (0.1%) and non-limiting(1.0%) concentrations of glucose. In the data presented in FIG. 6, atvarious times, the level of autoinducer produced in both the cell-freeculture fluids and in the corresponding 0.4M NaCl osmotic shock fluidswas measured and normalized for 1×10⁹ cells. It should be noted thatunlike in FIG. 5, the cells in this experiment were not pre-induced forsignal production.

[0212]FIG. 6 shows that the pattern of production and disappearance ofautoinducer observed in 0.4M NaCl osmotic shock fluids mimics thatobserved in cell-free culture fluids. However, at every time point thatautoinducer is produced, much greater activity is detected in theosmotic shock fluids than in the corresponding cell-free culture fluids.Under conditions of limiting (0.1%) glucose (FIGS. 6A, 6C and 6E), S.typhimurium produces the signaling activity between 2-4 h (Bars).However, the glucose becomes completely depleted at 4 h, and at thattime production of the factor ceases (FIG. 6A). In contrast, when thecells are grown in 1.0% glucose (FIGS. 6B, 6D, and 6F), glucose ispresent in the medium throughout the entire experiment (FIG. 6B). Underthese conditions, the cells continue to synthesize activity for 12hours. Similar to the results shown in FIG. 5 and those reported inExample 1, almost no activity was observed in cell-free culture fluidsor osmotic shock fluids from stationary phase cells at 24 h regardlessof the glucose concentration.

[0213]S. typhimurium grows at roughly the same rate in both high and lowglucose media during exponential phase. In fact, the S. typhimuriumculture grown in high glucose medium does not reach the cell densityachieved by the S. typhimurium grown in the low glucose medium (FIGS. 6Cand 6D). Cell growth is probably inhibited in this culture by thedramatically reduced pH that occurs from increased glucose utilization.These results show that the higher level of activity produced by S.typhimurium in the LB containing 1% glucose is not due to higher cellnumber, but due to induction of signal production caused by glucosemetabolism.

[0214]FIGS. 6E and 6F show the pH of the low and high glucose culturesat each time point. Under conditions of low glucose (FIG. 6E), the pH ofthe culture initially decreases as the cells utilize the glucose.However, simultaneous to the complete depletion of the glucose, the pHbegins to rise. In contrast, under conditions of high glucose, the pH ofthe medium decreases to below pH 5 (FIG. 6F). In the experimentspresented in FIG. 6, both glucose catabolism and decreasing pH occursimultaneously suggesting that either or both of these factors could beresponsible for signal production by S. typhimurium.

[0215] Both glucose metabolism and low pH independently control signalproduction in S. typhimurium. To distinguish between the contributionfrom glucose metabolism and that from low pH in signal production by S.typhimurium, we compared the activity produced by S. typhimurium grownin LB containing 0.5% glucose in a culture in which the pH wasmaintained at 7.2 (FIG. 7A), to that produced by S. typhimurium grown inLB without glucose where the pH was maintained at 5.0 (FIG. 7B). Again,we measured the signal present in cell-free culture fluids and in 0.4MNaCl osmotic shock fluids. Similar to the data presented in FIG. 3, thelevel of signal observed in cell-free culture fluids was lower than thatobserved in the 0.4M osmotic shock fluids.

[0216] When S. typhimurium was grown in LB+0.5% glucose at pH 7.2increasing amounts of the quorum sensing signal were detected for 6 h.At 6 h, in 0.4M NaCl osmotic shock fluids, there was an approximately550-fold stimulation of light production of the V. harveyi reporterstrain BB170. No activity was produced after the 6 h time point. FIG. 7Ashows that the pH was maintained between 7.15 and 7.25 for 8 h, afterthis time, the pH of the culture no longer decreased, but beganincreasing presumably because the cells had depleted the glucose. Weallowed the pH to continue to increase for the duration of theexperiment. Also shown in the Figure is the cell number at each timepoint. At pH 7.2, the cells grew rapidly and reached a high celldensity.

[0217] Analysis of time courses similar to those presented here, hasshown that S. typhimurium does not produce any signal when it is grownin LB without glucose at neutral pH (see Example 1). However, S.typhimurium did transiently produce the quorum sensing factor in theabsence of glucose when grown at pH 5.0 (FIG. 7B). Signal was producedfor 4 h, and about 450-fold stimulation of the reporter was the maximumactivity achieved in 0.4M NaCl osmotic shock fluids. Very little signalwas produced by 5 h, and signal was completely absent after 6 h ofincubation. FIG. 7B shows that the pH was maintained between 5.0 and 5.2in this experiment. Note that the cells grew much more slowly at pH 5.0than at pH 7.2.

[0218] Preliminary characterization of the S. typhimurium autoinducerdegradative apparatus. The quorum sensing activity produced by S.typhimurium LT2 is degraded by the onset of stationary phase. We havedetermined that the activity contained in cell-free culture supernatantsand 0.4M NaCl osmotic shock fluids from cells grown for 6 h inLB+glucose is stable for at least 24 h at 30° C., indicating that nodegradative activity is present in these cell-free fluids. Furthermore,mixing cell-free culture fluids prepared from actively producing S.typhimurium (i.e., from cultures grown for 6 h in LB+glucose) withcell-free culture fluids prepared from S. typhimurium that have degradedthe factor (i.e., from cultures grown for 12 or 24 h in LB+glucose) doesnot result in degradation of the activity. This result indicates thatthe degradative activity is not released, but instead, is associatedwith the cells.

[0219] We show in FIG. 5 that no further autoinducer is produced if S.typhimurium cells that are actively releasing autoinducer are shifted to0.1M NaCl. However, when these same cells are shifted to 0.4M NaCl, weobserve even greater autoinducer production. This result implies thatlow osmolarity could be a signal that induces the autoinducerdegradative machinery. To begin to analyze the mechanism by whichosmolarity affects autoinducer production and degradation in S.typhimurium, we investigated the requirement for protein synthesis insignal production and degradation by S. typhimurium in high and lowosmolarity. As described in the legend to FIG. 5, S. typhimurium LT2 wasgrown in LB containing 0.5% glucose to achieve maximal induction ofsignal production then treated with 0.1M or 0.4M NaCl in the presenceand absence of protein synthesis. Cell-free fluids were prepared andtested for signaling activity. Because half of the cell-free osmoticshock fluids contained chloramphenicol (Cm), V. harveyi JAF305 was usedas the reporter strain in the activity assay. This V. harveyi straincontains a Cm^(r) cassette in the luxN gene, and its phenotype is Sensor1⁻, Sensor 2⁺, a phenotype identical to that of V. harveyi BB170.

[0220] When the cells were resuspended in 0.4M NaCl, the S. typhimuriumproduced and released increasing amounts of the signal for 200 min (FIG.8A, open squares). After this time, the level of signaling activitypresent in the cell-free osmotic shock fluid decreased somewhat,suggesting that some of the released signal was degraded. Quitedifferent results were obtained when the S. typhimurium cells wereresuspended in 0.1M NaCl (FIG. 8B, open squares). In this case, at earlytime points, the S. typhimurium produced a quantity of activityequivalent to that produced by cells resuspended in 0.4M NaCl. However,by 120 min, no activity remained in the cell-free low osmolarity fluid.This result indicates that under conditions of low osmolarity, thereleased activity is rapidly degraded. We do not observe degradation ofthe activity in cell-free culture fluids, indicating that thedisappearance of the activity from low osmolarity cell-free fluids isnot due to chemical instability of the signaling molecule.

[0221] Under conditions of high osmolarity, when the cells were treatedwith Cm to inhibit protein synthesis, only about one quarter of theactivity was produced compared to untreated cells. The closed squares inFIG. 8A show that 300-fold induction of the reporter strain occurred inthe presence of Cm as compared to 1200-fold induction with the untreatedcells (FIG. 8A, open squares). When the S. typhimurium was resuspendedin low osmolarity (FIG. 8B), roughly three-quarters of the activityproduced in the absence of Cm (open squares) was produced in thepresence of Cm (closed squares). In the presence of Cm, the releasedactivity was not degraded by 300 min in high osmolarity and onlypartially degraded in low osmolarity.

[0222] To show that high osmolarity does not inhibit AI-2 signaldegradation, we added the activity contained in the 0.4M NaCl cell-freeosmotic shock fluids to S. typhimurium cells that had been resuspendedin 0.1M NaCl for two hours. As shown in FIG. 8, these are cells that candegrade the factor. Table 3 shows that these S. typhimurium cellsdegraded greater than 98% of the signaling activity while incubated athigh osmolarity. The table also shows that S. typhimurium cells that hadbeen incubated in 0.4M NaCl (these are cells that are actively producingthe signal) released no further activity when resuspended in the 0.1MNaCl incubation fluid obtained from the actively degrading cells.Furthermore, mixing active and inactive 0.4M and 0.1M cell-free osmoticfluids did not result in degradation of the activity in the 0.4M fluids.TABLE 2 High osmolarity induces release and low osmolarity inducesdegradation of the S. Typhimurium signaling factor. TreatmentFold-induction of luminescence 0.1 M NaCl activity^(a) 4 0.4 M NaClactivity^(a) 944 0.1 M cells + 0.4 M activity^(b) 17 0.4 M cells + 0.1 Mactivity^(c) 6

[0223] The LuxR homolog SdiA is not involved in response to the AI-2autoinducer. A gene homologous to luxR of V. fischeri has beenidentified in E. coli and S. typhimurium and is called sdiA. Two reportssuggest that in E. coli, SdiA modestly regulates the expression of thecell division locus ftsQAZ in response to a factor present in cell-freeculture fluids (Garcia-Lara et al., 1996, supra), and in response to afew homoserine lactone autoinducers (Sitnikov, et al., 1996, supra).Completion of the sequence of the E. coli genome shows that no LuxIhomologue exists in E. coli so the locus responsible for thebiosynthesis of the hypothesized soluble factor(s) has not beendetermined. Overexpression of SdiA in S. typhimurium has recently beenshown to influence the expression of several ORFs located on the S.typhimurium virulence plasmid (Ahmer, et al., 1998, supra). As in the E.coli studies, SdiA activity in S. typhimurium is proposed to bemodulated by an extracellular factor.

[0224] It was possible that the AI-2 autoinducer that we have beencharacterizing in S. typhimurium and E. coli acted through SdiA. Wetested whether AI-2 had an effect on genes regulated by SdiA in E. coliand S. typhimurium. In E. coli, we assayed an ftsQ1p2p-lacZ reporter,and in S. typhimurium we assayed an rck::MudJ fusion in both an sdiA⁺and sdiA⁻ background. We tested the effects of addition of LB, 0.4MNaCl, 0.4M NaCl osmotic shock fluids containing AI-2 activity from S.typhimurium LT2, E. coli O157, and 0.4M NaCl osmotic shock fluid from E.coli DH5. We have shown previously in Example 1 that DH5 does notproduce AI-2 activity under our growth conditions.

[0225] For the E. coli experiments we determined that MC4100 andMC4100/pMS209 (containing ftsQ1p2p in the incorrect orientation) had nomeasurable -galactosidase activity. The level of -galactosidase producedby MC4100/pMS207 (containing the ftsQ1p2p-lacZ fusion) was roughly 20-30Miller units, and this level of activity did not change under any of theconditions tested here. This level of activity of the fusion wascomparable to that reported previously (Sitnikov et al., 1996, supra;Garcia-Lara et al., 1996, supra). In the S. typhimurium SdiA studies,similar to Ahmer et al. (1998, supra), we obtained ˜30 Miller units ofrck::MudJ activity in the sdiA⁺ background and this level was reduced to10 units in the sdiA⁻ background. No change in -galactosidase productionoccurred following the addition of AI-2 from E. coli or S. typhimurium.These results indicate that, if an extracellular factor exists thatmodulates the activity of SdiA, under the conditions we have tested, itis not AI-2.

[0226] Discussion

[0227] Quorum Sensing in E. coli and S. typhimurium. We have developed aheterologous bio-assay that enables us to detect an extracellularsignaling factor produced by S. typhimurium. The factor mimics theaction of AI-2 of the quorum sensing bacterium V. harveyi, and it actsspecifically through the V. harveyi Signaling System 2 detector LuxQ.Results using lacZ fusions to the ftsQ and rck promoters indicate that,under our assay conditions, the AI-2 quorum sensing factor does notsignal to SdiA, at least with respect to regulation of these genes. TheAI-2 quorum sensing system is therefore involved in a different S.typhimurium and E. coli signal transduction pathway than the one(s)investigated previously.

[0228]S. typhimurium LT2 produces an amount of activity roughlyequivalent to that produced by V. harveyi. We observe approximately800-fold stimulation of the V. harveyi reporter strain BB170 uponaddition of 10% S. typhimurium cell-free culture fluids. The timing oflux induction and the shape of the response curve of V. harveyi to theS. typhimurium signal are indistinguishable from those of V. harveyiresponding to its own AI-2. Furthermore, we have been successful atpartially purifying both the V. harveyi AI-2 and the S. typhimuriumsignal molecule using identical purification procedures. These tworesults lead us to believe that the S. typhimurium signaling molecule isidentical to or very closely related to AI-2 of V. harveyi.

[0229] Growth Conditions Regulate Signal Production and Degradation inS. typhimurium. In this example, we further characterize the regulationof the signaling activity in S. typhimurium LT2. Accumulation ofsignaling activity in S. typhimurium culture supernatants is maximalduring mid-exponential phase when the cells are actively utilizingglucose in rich medium. Under these growth conditions, utilization ofglucose is accompanied by a rapid drop in pH of the culture. The resultsdemonstrate that either glucose metabolism or low pH is sufficient toinduce S. typhimurium LT2 to produce the quorum sensing factor,indicating that both glucose and acidity generate independent signalsfor autoinducer production. In the presence of glucose, when the pH isnot maintained, probably both the decreasing pH and the presence of anappropriate carbon source contribute to the regulation of quorum sensingin S. typhimurium. The results also show that production of theautoinducer ceases prior to stationary phase in the presence of glucoseat neutral pH and in the absence of glucose at low pH. Therefore, acombination of acidic conditions and the absence of glucose is notrequired to cue S. typhimurium to terminate production of autoinducer.

[0230] In addition to glucose, growth on several other carbohydratesalso induces production of the signaling activity. These include bothPTS (fructose, mannose, glucitol, and glucosamine) and non-PTS(galactose and arabinose) sugars. These findings eliminate an exclusiverole for the PTS in the regulation of autoinducer biosynthesis. When theS. typhimurium LT2 are grown on several other carbon sources (acetate,glycerol, citrate and serine) no significant accumulation of signalingactivity is observed. We have demonstrated in Example 1 that the signalis not any of a number of substances known to be secreted by S.typhimurium including the major products of mixed acid fermentation.Clearly, production of the signaling molecule is precisely regulated bythe cells and is favored under conditions of growth on preferredcarbohydrates for reasons that we do not yet understand. Identificationof the signaling molecule and cloning of the biosynthetic gene(s) willaid in a fuller understanding of the regulation process.

[0231] Results presented in this example show that, in contrast to otherquorum sensing systems, the S. typhimurium signal does not accumulate instationary phase. At least two competing processes contribute to thisregulation; autoinducer production and autoinducer degradation. In thisexample we are defining autoinducer production as an increase in thesignaling activity present in cell-free fluids. We recognize that anincrease in activity could result from release of newly biosynthesizedautoinducer, release of stored autoinducer, repression of degradation ofautoinducer, or some combination of these activities. We defineautoinducer degradation as the disappearance of signaling activity fromthe cell-free fluids. This disappearance could be due to destruction ofthe autoinducer, re-uptake of the autoinducer, or a combination of theseactivities. It is possible that under some of the conditions used in ourstudies, autoinducer production and autoinducer degradation areoccurring simultaneously. If this is the case, the activity detected incell-free culture fluids is a measure of which of these processes,production or degradation, predominates. These findings indicate thatquorum sensing in S. typhimurium is regulated such that the signal andpresumably the response to the signal do not persist into stationaryphase. Because the utilization of a preferred carbohydrate is alsorequired for signal production, quorum sensing in S. typhimurium may beused both for measuring the cell density and for measuring the potentialof the environment for growth.

[0232] Osmolarity Influences Signal Production and Degradation in S.typhimurium. S. typhimurium cells that are actively producing signal canbe further stimulated to produce signal by specific environmentaltreatments, indicating that several independent regulatory pathwayschannel information into autoinducer synthesis. One of these treatmentsis 0.4M NaCl osmotic shock. When autoinducer producing S. typhimuriumcells are resuspended in 0.4M NaCl, the cells release significantlygreater activity when they have the capability to synthesize proteinthan when protein synthesis is blocked. Furthermore, degradation of thesignal also requires protein synthesis. These results have severalimplications. First, in the presence of Cm, S. typhimurium resuspendedat both high and low osmolarity produce a similar amount of activity.This result indicates that, following growth in the presence of glucose,the S. typhimurium cells have a pre-defined capacity to producesignaling activity (and/or to release already synthesized activity fromthe cell). Second, when the cells are resuspended at high osmolarity,signal production increases well beyond this level. This increase insignal production requires protein synthesis, and we interpret this tomean that high osmolarity is one environmental cue that induces S.typhimurium to synthesize more of the biosynthetic apparatus necessaryfor signal production and/or release. Third, under conditions of lowosmolarity, we observe an initial release of activity, followed by arapid degradation of the activity. And, signal degradation requiresprotein synthesis because it is not observed in the presence of Cm.These results imply that the environment has changed from conditionsfavoring autoinducer production (LB+a preferred carbohydrate, or highosmolarity) to conditions where autoinducer production is not favored(low osmolarity, or absence of a preferred carbon source). Thisenvironmental change induces S. typhimurium to synthesize the protein(s)required for degradation of the signaling activity.

[0233] When the S. typhimurium cells were incubated in 0.4M NaCl nosignificant degradation of the activity occurred by 200 min. This resultindicates that either the necessary degradative protein(s) are notsynthesized under these conditions, or alternatively, the degradativeapparatus is assembled, but its activity is inhibited by highosmolarity. The results show that high osmolarity does not inhibitsignal degradation, because cells induced to degrade the activity can doso at high osmolarity. Therefore, the persistence of the activity in thehigh NaCl samples occurs because the degradation machinery is notsynthesized, not because its activity is inhibited.

[0234] It is difficult to precisely determine when S. typhimurium cellsare autoinducer producers and when they are autoinducer degradersbecause both processes could occur simultaneously. It appears, however,that no or very low degradation occurs in high osmolarity, andconversion of cells from overall signal producers to overall signaldegraders occurs in low osmolarity and requires protein synthesis. Ourpreliminary characterization of the degradative process indicates thatit is cell-associated because the autoinducer activity is stable incell-free culture supernatants for long periods of time, and combiningactive with inactive cell-free culture fluids or active and inactivehigh and low osmolarity cell-free fluids does not promote degradation ofthe autoinducer. We have recently isolated a S. typhimurium mutant thatdoes not produce the AI-2 activity. If this mutant retains thecapability to degrade autoinducer, analysis of it will be informativefor understanding the timing of degradation, and for identifying thecues that induce the degradative machinery. We are currently attemptingto isolate S. typhimurium mutants capable of autoinducer production butincapable of autoinducer degradation.

[0235] The Role for Quorum Sensing in Salmonella Pathogenesis. Theobservations presented here on the regulation of signal production anddegradation by S. typhimurium LT2 implicate a role for quorum sensing inpathogenesis of Salmonella. The conditions favoring signal production(nutrient rich, high osmolarity and low pH) are those likely to beencountered upon the first interaction of an enteric pathogen with itshost. Conditions favoring degradation of the signal (nutrient poor, lowosmolarity) are those most probably encountered as the pathogen exitsthe host. The initial colonization of the host may be a concerted effortbetween a population of cells coordinated through this cell-cellsignaling system. Other cues, that we have not yet tested, could alsoregulate quorum sensing in S. typhimurium. These may representindependent or overlapping signaling pathways involved in pathogenesis.We are isolating S. typhimurium mutants to test these hypotheses.Finally, Salmonella pathogenesis is a dynamic process of interactionbetween the host and metabolically active bacteria. Consistent with arole for quorum sensing in pathogenesis, our evidence suggests that thisquorum sensing system is not functioning during stationary phase. Wehave shown that the signaling molecule is not produced during stationaryphase, and furthermore, existing signal is degraded. Perhaps quorumsensing is critical for S. typhimurium to undergo the transition betweena host-associated and a free-living existence.

EXAMPLE 3 Quorum Sensing in Escherichia coli, Salmonella typhimurium andVibrio harveyi: A New Family of Genes Responsible for AutoinducerProduction

[0236] In this example we report the analysis of a gene responsible forAI-2 production in V. harveyi, E. coli and S. typhimurium. The geneidentified in all three species of bacteria is highly homologous, and wepropose that these genes define a new family of proteins involved inautoinducer production. The genes, which we named luxS_(V.h.),luxS_(E.c.), and luxS_(S.t.) have been identified in many species ofbacteria by genome sequencing projects, but until now no function hasbeen ascribed to this gene in any organism. The luxS genes do not bearhomology to any other gene known to be involved in autoinducerproduction.

[0237] Materials and Methods

[0238] Bacterial strains, media and recombinant DNA techniques. V.harveyi BB120 is the wild type strain (Bassler et al., 1997, supra). S.typhimurium strain LT2 was obtained from Dr. K. Hughes (University ofWashington), S. typhimurium 14028 is ATCC strain 14028 Organism:Salmonella choleraesuis. E. coli O157:H7 is a clinical isolate suppliedby Dr. Paddy Gibb (University of Calgary). Luria broth (LB) contained 10g Bacto Tryptone (Difco), 5 g Yeast Extract (Difco) and 10 g NaCl perliter. The recipe for Autoinducer Bioassay (AB) medium has been reportedpreviously (Greenberg, E. P., Hastings, J. W., and Ulitzur, S. (1979)Arch. Microbiol. 120, 87-91). Where specified, glucose was added from asterile 20% stock to a final concentration of 0.5%. Antibiotics wereused at the following concentrations (mg/L): Ampicillin (Amp) 100,Chloramphenicol (Cm) 10, Gentamycin (Gn) 100, Kanamycin (Kn) 100, andTetracycline (Tet) 10. DNA isolation, restriction analysis andtransformation of E. coli was performed as described by Sambrook et al.Probes for Southern Blot analysis were labeled using the Multiprime DNAlabeling system of Amersham. Sequencing was carried out using an AppliedBiosystems sequencing apparatus. The V. harveyi BB120 genomic librarywas constructed in the cosmid pLAFR2 as described (Bassler et al., 1993,supra). The method for Tn5 mutagenesis of cloned V. harveyi genes, andthe allelic replacement technique for inserting Tn5 mutated genes intothe V. harveyi chromosome have been reported (Bassler et al., 1993,supra).

[0239] Bioluminescence Assay. The AI-2 bioassay using the V. harveyireporter strain BB170 (Sensor 1⁻, Sensor 2⁺) has been discussed in theprevious examples. Cell-free culture fluids from V. harveyi, E. coli, orS. typhimurium strains to be tested for AI-2 activity were prepared asdescribed above, and assayed at 10% (v/v). AI-2 activity is reported asthe fold-induction of the reporter strain over background, or as thepercent of the activity obtained from V. harveyi BB120 (wild type)cell-free culture fluid.

[0240] Mutagenesis and analysis of the AI-2 production gene in S.typhimurium LT2. MudJ insertion mutants of S. typhimurium LT2 weregenerated using a phage P22 delivery system as described (Maloy, S. R.,Stewart, V. J., and Taylor, R. K. (1996) Genetic analysis of pathogenicbacteria: a laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). Following growth to mid-exponential phase in LBcontaining 0.5% glucose, the S. typhimurium insertion mutants weretested for AI-2 production using the V. harveyi BB170 bioassay. The siteof the MudJ insertion that inactivated the AI-2 production function inS. typhimurium was identified by PCR amplification and sequencing of thechromosomal DNA at the insertion junction. A two-step amplificationprocedure was used (Caetano-Annoles, G. (1993) Meth. Appl. 3, 85-92). Inthe first PCR reaction, the arbitrary primer5′-GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCCC-3′, and the MudJ specific primer5′-GCACTACAGGCTTGCAAGCCC-3′ were used. Next, 1 l of this PCR reactionwas used as the template in a second PCR amplification employing asecond arbitrary primer (5′-GGCCACGCGTCGACTAGTCA-3′) and another MudJspecific primer (5′-TCTAATCCCATCAGATCCCG-3′). The PCR product from thesecond reaction was purified and sequenced.

[0241] Cloning and sequencing of the E. coli MG1655, E. coli O157:H7,and E. coli DH5 AI-2 production genes. The DNA sequence obtained fromthe S. typhimurium LT2 MudJ screen was used to search the E. coli MG1655genome sequence to identify the corresponding E. coli region (Blattneret al., Science 277, 1453-1462, 1997). The gene identified from thesequencing project had the designation ygaG. Primers that flanked theygaG gene and incorporated restriction sites were designed and used toamplify the E. coli MG1655, E. coli O157:H7 and E. coli DH5 ygaG genes.The primers used are: 5′-GTGAAGCTTGTTTACTGACTAGATC-3′ and5′-GTGTCTAGAAAAACACGCCTGACAG-3′. The PCR products were purified,digested and cloned into pUC19. In each case, the PCR products fromthree independent reactions were cloned and sequenced.

[0242] Results

[0243] Identification and cloning of the gene responsible for AI-2production in V. harveyi. We have discussed in previous examples that,unlike many other E. coli strains, E. coli strain DH5 does not producean AI-2 signal molecule that can be detected by V. harveyi. We reasonedtherefore, that we could use E. coli DH5 as a mutant to clone the V.harveyi AI-2 production gene. A library of wild type V. harveyi BB120genomic DNA was transformed into E. coli strain DH5, and thetransformants were screened for AI-2 production in the V. harveyi BB170AI-2 detection bioassay. The library consisted of 2,500 clones eachcontaining roughly 25 kb of V. harveyi genomic DNA. Five DH5 clones wereidentified that resulted in upwards of 300-fold stimulation of thereporter strain in the bioassay.

[0244] The recombinant cosmid DNA from the five AI-2 producing E. coliDH5 clones was analyzed by restriction analysis and Southern blotting.All five of the cosmids contained an overlapping subset of identical V.harveyi genomic restriction fragments, indicating that we had cloned thesame locus several times. One cosmid, called pBB2929 was selected forfurther analysis. Random mutagenesis using transposon Tn5 was carriedout on cosmid pBB2929, and pools of cosmids harboring Tn5 insertionswere subsequently transformed into E. coli DH5. We tested 962 individualE. coli DH5/pBB2929::Tn5 strains for the loss of the ability to produceAI-2. Four E. coli DH5 strains harboring Tn5 insertions in pBB2929 wereidentified that failed to produce AI-2. We mapped the locations of theseTn5 insertions in pBB2929 and found that all four transposon insertionsresided in the same 2.6 kb HindIII V. harveyi genomic DNA fragment (FIG.9A).

[0245] Cosmid pBB2929 was digested with HindIII and the 8 resultingfragments were subcloned in both orientations into pALTER (Promega). ThepALTER subclones were transformed into E. coli DH5, and subsequentlytested for AI-2 production. The only strains capable of producing AI-2contained the 2.6 kb HindIII fragment identified in the Tn5 mutagenesis.This fragment was sequenced, and only one open reading frame (ORF) couldbe identified, and its location corresponded to the map positions of thefour Tn5 insertions that eliminated AI-2 production. We named the ORFluxS_(V.h.) (FIG. 9A).

[0246] Mutagenesis of luxS_(V.h.) in V. harveyi. We analyzed the effectsof luxS_(V.h.) null mutations on AI-2 production in V. harveyi. The fourTn5 insertions that mapped to the luxS_(V.h.) gene and the control Tn5insertion adjacent to the luxS_(V.h.) locus were transferred to thecorresponding locations in the V. harveyi BB120 chromosome to makestrains MM37, MM30, MM36, MM38 and MM28 respectively (FIG. 9A). Southernblotting was used to confirm the correct placement of all five Tn5insertions in the V. harveyi chromosome. The four V. harveyiluxS_(V.h.)::Tn5 insertion strains were tested for the ability toproduce AI-2, and all four strains gave identical results.

[0247] In FIG. 10A, we show the AI-2 production phenotypes of the wildtype control Tn5 insertion strain MM28 and one representativeluxS_(V.h.)::Tn5 insertion strain, MM30. V. harveyi MM28 and MM30 weregrown to high cell density, after which cell-free culture fluids wereprepared. The culture fluids were assayed for AI-2 activity by theability to induce luminescence in the AI-2 detector strain BB170. FIG.10A shows that addition of culture fluids from the control Tn5 insertionstrain MM28 induced luminescence in the reporter 780-fold, while culturefluid from the luxS_(V.h.)::Tn5 insertion strain MM30 did not induce theexpression of luminescence in the reporter. Therefore, a null mutationin luxS_(V.h.) in V. harveyi eliminates AI-2 production.

[0248] Identification and analysis of S. typhimurium autoinducerproduction mutants. In order to identify the gene responsible for AI-2production in S. typhimurium, we randomly mutagenized S. typhimurium LT2using the MudJ transposon (Maloy et al., 1996, supra). Ten-thousand S.typhimurium LT2 insertion mutants were assayed for AI-2 production inthe V. harveyi BB170 bioassay. One S. typhimurium MudJ insertion mutant(strain CS132) was identified that lacked detectable AI-2 in culturefluids at mid-exponential phase.

[0249]FIG. 10B shows the AI-2 production phenotypes of S. typhimuriumstrain LT2 and the corresponding MudJ insertion strain CS132. Thestrains were grown to mid-exponential phase in LB containing glucose,and cell-free culture fluids were prepared and assayed for AI-2. S.typhimurium LT2 culture fluids induced the reporter strain 500-fold,while culture fluids from strain CS132 contained no AI-2 activity.Furthermore, strain CS132 did not produce AI-2 under any of the growthconditions that we have previously reported induce AI-2 production in S.typhimurium (not shown).

[0250] The site of the MudJ insertion in S. typhimurium CS132 wasdetermined by PCR amplification followed by sequencing of the 110 bp ofchromosomal DNA adjacent to the transposon. This sequence was used tosearch the database for DNA homologies. The sequence matched a site(89/105 bp identity) in the E. coli MG1655 genome that corresponded toan ORF of unknown function denoted ygaG (Blattner et al., 1997, supra).In the chromosome, the E. coli ygaG gene is flanked by the gshA and emrBgenes (FIG. 9B). The ygaG gene is transcribed from its own promoterwhich is located immediately upstream of the gene, indicating that it isnot in an operon with gsha. The emrB gene is transcribed in the oppositedirection. We PCR amplified the ygaG region from the chromosomes of E.coli O157:H7 and E. coli MG1655, and the two E. coli ygaG genes werecloned into pUC19.

[0251] Complementation of S. typhimurium and E. coli AI-2-mutants. Wetested whether the E. coli O157:H7 ygaG gene and the V. harveyiluxS_(V.h.) gene could restore AI-2 production in the AI-2 strains S.typhimurium CS132 and E. coli DH5. In FIG. 11A, we show the AI-2activity produced by wild type V. harveyi BB120, E. coli O157:H7 and S.typhimurium LT2. In this figure, the level of AI-2 activity present inV. harveyi BB120 cell-free culture fluids was normalized to 100%, andthe activities in cell-free culture fluids from E. coli and S.typhimurium compared to that. In this experiment, E. coli O157:H7produced 1.5 times and S. typhimurium LT2 produced 1.4 times more AI-2activity than V. harveyi BB120 (i.e., 150% and 141% respectively).

[0252]FIGS. 11B and 11C show the AI-2 complementation results for S.typhimurium CS132 and E. coli DH5. FIG. 11B demonstrates thatintroduction of the E. coli O157:H7 ygaG gene into S. typhimurium CS132restored AI-2 production beyond the level of production of wild type S.typhimurium (i.e., 209% activity). Comparison of the data in FIGS. 11Aand 11B shows that the E. coli ygaG gene in S. typhimurium resulted inAI-2 production exceeding that produced in vivo by E. coli O157:H7.Introduction of the V. harveyi luxS_(V.h.) gene into S. typhimuriumresulted in AI-2 production at slightly less than the level produced bywild type V. harveyi BB120 (i.e., 73% of the level of V. harveyi BB120).FIG. 11C shows that E. coli DH5 was also complemented to AI-2 productionby both the cloned E. coli O157:H7 and the V. harveyi BB120 AI-2production genes. However, introduction of E. coli O157:H7 ygaG and V.harveyi BB120 luxS_(V.h.) into E. coli DH5 resulted in only 31% and 43%of the V. harveyi BB120 AI-2 activity respectively. FIGS. 11B and 11Cshow that the control vectors produced no activity in thecomplementation experiments.

[0253] Analysis of the AI-2 production genes from V. harveyi, E. coliand S. typhimurium. We sequenced the AI-2 production gene luxS_(V.h.)from V. harveyi BB120 and the ygaG loci from E. coli O157:H7, E. coliMG1655 and E. coli DH5. The translated protein sequences encoded by theygaG ORF's are shown in FIG. 12, and they are aligned with thetranslated LuxS protein sequence from V. harveyi. The non-bold,underlined amino acids indicate the residues in the E. coli proteinsthat differ from the V. harveyi LuxS protein. The ygaG loci from E. coliencode proteins that are highly homologous to one another and also toLuxS from V. harveyi. The E. coli MG1655 and the E. coli O157:H7 YgaGproteins are 77% and 76% identical to LuxS from V. harveyi BB120. TheDNA sequence we determined for ygaG from E. coli O157:H7 differs at fivesites from the reported (and our) sequence for the E. coli MG1655 ygaGgene. Four of the changes are silent, the fifth results in aconservative Ala to Val alteration at amino acid residue 103 in the E.coli O157:H7 protein.

[0254] Identification of the ygaG locus in E. coli MG1655 and E. coliO157:H7 allowed us to investigate the AI-2 production defect in E. coliDH5. E. coli DH5 possesses the ygaG gene because we could PCR amplifythis region from the chromosome using the same primers we employed toamplify it from E. coli MG1655 and E. coli O157:H7. Examination of theE. coli DH5 ygaG promoter showed that it is identical to that of E. coliMG1655, indicating that the AI-2 defect in E. coli DH5 is not simply dueto decreased transcription of ygaG. However, sequence analysis of the E.coli DH5 ygaG coding region showed that a one G-C base pair deletion anda T to A transversion exist at bp 222 and 224 respectively. Theframeshift mutation resulting from the G/C deletion causes prematuretruncation of the E. coli DH5 protein. FIG. 12 shows that the truncatedE. coli DH5 protein is 111 amino acids, while the E. coli MG1655 and E.coli O157:H7 proteins are 171 residues. Twenty altered amino acids aretranslated after the frame shift and prior to termination of theprotein. Our complementation results (FIG. 11) demonstrate that the AI-2production defect in E. coli DH5 is recessive to in trans expression ofygaG, which is consistent with the defect being due to a null mutationcaused by the frame shift in the E. coli DH5 ygaG gene.

[0255] We searched the S. typhimurium database using the sequence weobtained adjacent to the MudJ that inactivated the AI-2 productionfunction in S. typhimurium CS132. A perfect match (110/110 bp) wasidentified to fragment B_TR7095.85-T7 in the S. typhimurium LT2 genomesequencing database (Genome Sequencing Center, Washington University,St. Louis). However, the S. typhimurium LT2 database ygaG sequence isincomplete (FIG. 12). The translated sequence matches the E. coli and V.harveyi sequences beginning at amino acid residue 8. The translatedsequence shows that the S. typhimurium protein is 75% identical to LuxSof V. harveyi. In order to align the S. typhimurium sequence with the V.harveyi LuxS protein, we corrected three apparent frame shift errors inthe database sequence. Considering that only crude, unannotated sequencedata is currently available for S. typhimurium, we predict that the S.typhimurium protein contains 7 more amino acids, and that the frameshift mutations are sequencing errors. We were unsuccessful at PCRamplifying either the S. typhimurium 14028 or the S. typhimurium LT2ygaG gene using the primers designed for E. coli, so we do not yet havethe complete sequence of the S. typhimurium gene.

[0256] The results set forth above indicate that the genes we haveidentified and analyzed encode a novel family of proteins responsiblefor autoinducer production. Members of this new family of genes,referred to herein as LuxS, are highly homologous to one another but notto any other identified genes. The encoded product of the LuxS genescatalyze an essential step in the synthesis of the signaling moleculesof the present invention.

EXAMPLE 4 Construction of a Sensor 1⁻, AI⁻2⁻ V. harveyi Reporter Strain

[0257]V. harveyi null mutants in each of the lux genes luxL, luxM, luxN,luxS and luxQ have been constructed. These mutants fail to either makeor respond to one specific autoinducer, but they still produce lightbecause, in each case, one quorum sensing system remains operational. Adouble luxN, luxS V. harveyi mutant will not emit light without theaddition of exogenous AI-2 because this mutant will not respond to AI-1and it will not produce AI-2.

[0258] The V. harveyi luxS gene has been cloned into E. coli DH5α on abroad host range mobilizable cosmid called pLAFR2. This constructionrestores AI-2 production to E. coli DH5α. A marked null mutation wasengineered into the luxS gene by introducing a chloramphenicolresistance (Cm^(r)) cassette into an internal restriction site.Placement of the Cm^(r) cassette at this site in luxS subsequentlyeliminated AI-2 production in E. coli DH5α.

[0259] The luxS::Cm^(r) null allele was transferred onto the chromosomeof V. harveyi strain BB170. Strain BB170 contains a Tn5Kan^(r) in luxNand does not respond to AI-1. To construct the double mutant,triparental conjugations were carried out by mixing stationary phasecultures of E. coli DH5α carrying the V. harveyi luxS::CM^(r)construction in pLAFR2 (pLAFR2 carries tetracycline resistance), E. coliDH5a carrying the tra donor plasmid pRK2013 and the V. harveyi recipientstrain BB170. Exchange of the luxS::Cm^(r) mutant allele for the wildtype luxS allele on the chromosome occurs by homologous recombination.The exogenote cosmid in V. harveyi was eliminated by the introduction ofa second incompatible plasmid pPH1JI. This was accomplished by mating E.coli DH5α containing pPH1JI with the V. harveyi BB170 recipientcontaining the luxS::Cm^(r) cosmid, and selecting for exconjugants onplates containing ampicillin (for counter selection of the E. colidonor) chloramphenicol (for inheritance of the mutant luxS::Cm^(r)allele) and gentamicin (for maintenance of the plasmid pPH1JI). Southernblot analysis was used to verify that the exogenote pLAFR2 cosmid hasbeen eliminated and that the luxS::Cm^(r) construction had beentransferred to the corresponding position in the genome of V. harveyi.The pPH1JI cosmid was subsequently eliminated by growth in the absenceof gentamicin selection.

[0260] Verification that the luxN, luxS Double Mutant Responds to AI-2.The V. harveyi strain that is mutant in luxN and luxS was stimulated toproduce light in response to the exogenous addition of AI-2 but notAI-1. This was verified in a luminescence assay for response to V.harveyi AI-1 and AI-2. V. harveyi strain MM30 (luxS::Tn5) which isphenotypically AI-1⁺, AI-2⁻, and V. harveyi strain BB152 (luxM::Tn5)which is phenotypically AI-1⁻, AI-2⁺ were used as the sources of AI-1and AI-2 respectively. The AI-1 and AI-2 present in culture fluids ofthese strains was tested for stimulation of light production of the V.harveyi luxN, luxS double mutant reporter strain. In this assay,autoinducer preparations from MM30, BB152 or sterile medium controlswere added to the wells of microtiter plates, followed by the additionof the V. harveyi reporter strain. The resulting light production wasmonitored using a liquid scintillation counter in the chemiluminescencemode. Maximal stimulation of light production in the V. harveyi luxN,luxS reporter strain was compared to that produced by the Sensor 1⁺,Sensor 2⁻ V. harveyi strain BB886 and the Sensor 1⁻, Sensor 2⁺ V.harveyi strain BB170. These two V. harveyi strains are routinely used inthis assay as reporters of AI-1 and AI-2 activity respectively.

[0261] Determine optimum concentrations of AI-2 in microtiter assays.The aformentioned screen will be optimized for use in 96-well microtiterassays. The screen will be used in inhibitor assays for identifyinginhibitors of AI-2. Purified or synthetic AI-2 will be added to themicrotiter wells containing the newly constructed reporter strain andinhibition will be measured by a decrease in light emission from thewells containing an inhibitor. The assay will be optimized bydetermining the concentration of cells and AI-2 in the microtiter wellsthat will allow for maximal sensitivity. The optimal AI-2 concentrationwill be that which stimulates half-maximal light output for a givenconcentration of cells per unit time. Initial experiments will beconducted in this concentration range to determine the range of AI-2concentration that produces the greatest change in light output. Similarexperiments using AI-1 and a non self-stimulating sensor-1⁺, sensor-2⁻mutant (BB886) showed that the assay was sensitive to concentrations aslow as 100 nM AI-1 and that light emission was linear over 6 orders ofmagnitude (light emission from a self-stimulating strain was linear over3 orders). Similar results for AI-2 using the new reporter strain whichwill not self-stimulate and therefore have zero background lightemission are predicted. Light emission from the microtiter wells will bemeasured with a Wallac TriLux liquid scintillation counter model1450-021 in the chemiluminescence mode. This machine will accommodate 16plates and will therefore allow for 1536 separate concentrationexperiments per run.

EXAMPLE 5 In-vitro Method for Synthesizing AI-2

[0262] Purification and Identification of AI-2. The AI-2 class ofmolecule is refractory to purification by conventional techniques usedfor the isolation of acyl-homoserine lactone (HSL) autoinducers such asAI-1 from V. harveyi. Unlike other HSL autoinducers, under theconditions tested, the AI-2 activity does not extract quantitativelyinto organic solvents. Furthermore, it fails to bind to either a cationor anion exchange column. The present characterization of AI-2 indicatesthat it has a molecular weight of less than 1000 kDa, and is a polar butapparently uncharged organic compound. The AI-2 activity is acid stableand base labile and heat resistant to about 80 but not 100° C. Theseresults indicate that the AI-2's are not acyl-homoserine lactones. TheluxS genes identified in the present study bear no homology to othergenes known to be involved in production of HSL autoinducers furtherindicating that the present AI-2 class of autoinducers is novel.

[0263] Thus, in addition to providing a cloned, overexpressed andpurified S. typhimurium LuxS protein, the present invention alsoprovides a method for producing AI-2 in vitro. The present inventionprovides a mechanism for generating large quantities of pure AI-2 usefulfor mass spectral and NMR analysis, and for screening compounds whichmodulate the activity of AI-2. Moreover, the present invention providesa method for determining the in vivo biosynthetic pathway for AI-2synthesis.

[0264] The analysis of the genomic locations of the various luxS genesidentified in the present invention indicates that the luxS genes do notconsistently reside in any one location in the chromosome, nor are theytypically found in close proximity to any specific gene(s). However, inone case, the luxS gene is the third gene in a three-gene operon withtwo genes (metK and pfs). In E. coli, Salmonella and many otherbacteria, MetK and Pfs are involved in the conversion of S-adenosylmethionine (SAM) to homocysteine and 4,5-dihydroxy-2,3 pentanedione(FIG. 15). The function of MetK is to convert methionine to SAM which isan important cofactor in one-carbon metabolism. SAM is used to methylateDNA, RNA and a variety of cell proteins, and several SAM dependentmethyl transferases act at this step. S-adenosyl homocysteine (SAH) isproduced when the methyl group is transferred from SAM to itssubstrates. SAH functions as a potent inhibitor of SAM dependentmethyltransferases. Therefore, bacteria rapidly degrade SAH via theenzyme Pfs. The designation “pfs” refers to an open reading frame in theE.coli genome that has recently been determined to encode the enzyme5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase, also knownas MTA/SAH nucleosidase. In the present system, the enzyme cleaves theglycosidic bond in S-adenosylhomocycteine (SAH). Thus, the function ofPfs is to convert SAH to adenine and S-ribosyl homocysteine. In a finalstep, S-ribosyl homocysteine is converted to homocysteine and4,5-dihydroxy-2,3-pentanedione. Homocysteine can re-enter this pathway;it is methylated to generate methionine which can be converted to SAM byMetK.

[0265] The catabolism of SAH is considered a salvage pathway forrecycling metabolic intermediates (adenine and homocysteine). However,some species of bacteria eliminate SAH by a different pathway. In thisalternative pathway, adenosine is directly removed from SAH whichgenerates homocysteine. Therefore, cells that use this second mechanismdo not produce 4,5-dihydroxy-2,3-pentanedione. In the pathway shown inFIG. 15, the enzyme responsible for conversion of S-ribosyl homocysteineto 4,5-dihydroxy-2,3-pentanedione has never been identified, cloned orpurified. Furthermore, no role for 4,5-dihydroxy-2,3-pentanedione isknown.

[0266] LuxS is involved in the pathway shown in FIG. 15, and SAM and SAHare involved in AI-2 production. The structure of AI-2 could be4,5-dihydroxy-2,3-pentanedione, in which case LuxS is theuncharacterized enzyme that acts on S-ribosyl homocysteine. Second, LuxScould act on one of the intermediates to make AI-2. LuxS would representa branch point off the known pathway.

[0267] To confirm that LuxS is involved in the conversion of SAM toAI-2, the gene encoding the S. typhimurium LuxS protein was cloned,overexpressed and the S. typhimurium LuxS protein was purified. Thisprotein was used in combination with dialyzed cell-free extractsprepared from a S. typhimurium luxS null mutant to show that addition ofSAM and LuxS protein could restore AI-2 production to dialyzed LuxS-cellextracts. Reaction mixtures were prepared containing 10 mM SodiumPhosphate buffer pH 7.0, dialyzed S. typhimurium LuxS-cell extract andSAM. Purified LuxS protein was added to some of these mixtures. Thereactions were incubated at room temperature for 60 min, followed bycentrifugation in a 5000 MWCO centricon. The material with MW<5000 wasadded to the standard V. harveyi bioassay as previously described.Dialyzed LuxS-cell extracts to which SAM was added or extractscontaining LuxS protein without the addition of SAM produced no AI-2activity. However, identical extracts to which LuxS protein and SAM hadbeen added produced AI-2 that resulted in over 500-fold stimulation inlight production in the bioassay.

[0268] Further investigation showed that SAM is not the direct substratefor LuxS, and that LuxS must act at a step subsequent to the conversionof SAM to SAH (FIG. 15). It was determined that AI-2 was not produced ifSAM was added directly to LuxS protein, however activity was produced bypre-incubation of SAM with the LuxS-extracts, filtration, and subsequentaddition of LuxS protein to the filtrate. Importantly, these studiesindicate that SAM can react with an element in the cell extract beforeit can be used by LuxS to make AI-2. Presumably, the SAM dependentmethyl transferases present in the cell extract use SAM as a methyldonor and convert it to SAH in the process. To verify this, SAH wassubstituted for SAM in an in vitro assay. Addition of SAH to the invitro assay resulted in much greater AI-2 production than when SAM wasadded. This result indicates that LuxS functions in the pathwaysubsequent to the conversion of SAM to SAH. Again, addition of SAHdirectly to LuxS protein is not sufficient for production of AI-2activity, while pre-incubation of SAH with dialyzed LuxS-extractsfollowed by filtration and subsequent addition of LuxS protein to thefiltrates does result in AI-2 production. Presumably SAH is converted toS-ribosyl homocysteine and then LuxS acts to produce AI-2.

[0269] The proposed pathway shown in FIG. 15 is not a salvage pathwayfor recycling secondary metabolites, but rather is the pathway forproduction of AI-2. The present invention has narrowed the possibilitiesfor point of LuxS activity in the biosynthesis of AI-2. The remainingpossibilities are shown in FIG. 15 (designated LuxS?).

[0270] According to the invention, AI-2 is a deravitive of ribose. It isnoteworthy that, in V. harveyi, LuxP, the primary sensor for AI-2, is ahomologue of the E. coli and S. typhimurium ribose binding protein.

[0271] Characterization and Biosynthesis of AI-2. The invention furtherprovides a method for an in vitro procedure for large scale productionof pure AI-2. As indicated in FIG. 15, SAH is a precursor in the LuxSdependent biosynthesis of AI-2. Furthermore, LuxS does not act directlyon SAH. Prior to the action of LuxS, SAH must first be acted on by someenzyme in dialyzed cell extracts. Presumably this step is the conversionof SAH to S-ribosyl homocysteine by Pfs. Therefore the substrate forLuxS is S-ribosyl homocysteine.

[0272] To confirm that LuxS acts on S-ribosyl homocysteine, the Pfsenzyme can be purified and used to convert SAH to S-ribosylhomocysteine. Toward this end, the pfs gene has been cloned from S.typhimurium 14028 placed into the overexpression vector pLM-1. The Pfsenzyme will be overexpressed and SAH will be added to purified Pfs toproduce S-ribosyl homocysteine. The conversion of SAH to S-ribosylhomocysteine will be confirmed by reverse phase HPLC analysis (SAH is UVactive while S-ribosyl homocysteine is not). Subsequently, the S-ribosylhomocysteine produced by Pfs will be added to purified LuxS. Followingincubation, the mixture will be filtered over a 5000 MWCO centricon. Thefiltrate will be tested for AI-2 activity in the previously described V.harveyi bioassay. The identification of activity will confirm that4,5-dihydroxy-2,3-pentanedione is AI-2.

[0273] In addition, AI-2 structure obtained from E. coli and V. harveyiAI-2 will be determined. The E. coli and V. harveyi luxS genes have beencloned in to overexpression vectors. The identity/biosynthesis of the S.typhimurium AI-2 provided by the present invention should greatlyfacilitate these analyses. In the event that the S. typhimurium, E. coliand V. harveyi AI-2's are identical these data will indicate that AI-2'sare the same.

[0274] The present invention is not limited to the embodiments describedand exemplified above, but is capable of variation and modificationwithin the scope of the appended claims.

1 17 1 519 DNA Vibrio harveyi 1 atgcctttat tagacagctt taccgtagaccacacgcgta tgaatgcacc agcggttcgt 60 gtggctaaaa cgatgcaaac tccaaaaggagacaccatca cggtattcga cctacgtttc 120 actgctccaa acaaagacat cctttctgagaaaggaattc atacattaga gcatttgtac 180 gcaggcttta tgcgtaatca cctaaatggtgatagcgttg agatcattga tatctcacca 240 atggggtgcc gtactggttt ctacatgagcttgattggta cgccttcaga gcagcaagtg 300 gctgacgctt ggattgccgc gatggaagacgtactaaaag tagaaaacca aaacaagatc 360 cctgagttga acgaatacca atgtggtacagcagcgatgc actctctgga tgaagcgaag 420 caaatcgcga agaacattct agaagtgggtgtggcggtga ataagaatga tgaattggca 480 ctgccagagt caatgctgag agagctacgcatcgactaa 519 2 516 DNA Escherichia coli 2 atgccgttgt tagatagcttcacagtcgat catacccgga tggaagcgcc tgcagttcgg 60 gtggcgaaaa caatgaacaccccgcatggc gacgcaatca ccgtgttcga tctgcgcttc 120 tgcgtgccga acaaagaagtgatgccagaa agagggatcc ataccctgga gcacctgttt 180 gctggtttta tgcgtaaccatcttaacggt aatggtgtag agattatcga tatctcgcca 240 atgggctgcc gcaccggtttttatatgagt ctgattggta cgccagatga gcagcgtgtt 300 gctgatgcct ggaaagcggcaatggaagac gtgctgaaag tgcaggatca gaatcagatc 360 ccggaactga acgtctaccagtgtggcact taccagatgc actcgttgca ggaagcgcag 420 gatattgcgc gtagcattctggaacgtgac gtacgcatca acagcaacga agaactggca 480 ctgccgaaag agaagttgcaggaactgcac atctag 516 3 110 DNA Salmonella typhimurium misc_feature(0)...(0) sequence from MudJ 3 gatgtgctga aagtgcagga tcaaaaccagatcccggagc tgaacgttta ccagtgcggt 60 acgtatcaga tgcactcgct cagtgaagcgcaggacattg cccgtcatat 110 4 492 DNA Salmonella typhimurium misc_feature(0)...(0) sequence from alignment; starts with codon 8 and requires 3frameshifts 4 aattcggatc ataccggatg caagcgccgg cggtccgggt tgcaaaaacgatgaacaccc 60 cgcatggcga cgcaatcacg tgtttgatct gcgtttttgc attccgaacaaagaagtgat 120 gccggaaaaa gggattcata cgcttgagca tctgtttgct ggctttatgcgcgaccacct 180 caacggtaac ggcgttgaga ttatcgatat ctcgccgatg ggctgccgcaccggctttta 240 catgagcctg attggcacgc cggacgagca gcgtgttgcc gacgcctggaaagcggcgat 300 ggcggatgtg ctgaaagtgc aggatcaaaa ccagatcccg gagctgaacgtttaccagtg 360 cggtacgtat cagatgcact cgctcagtga agcgcaggac attgcccgtcatattctgga 420 gcgtgatgtg cgcgtgaaca gcaataaaga gctggcgctg ccgaaagaaaaactgcagga 480 actgatattt ag 492 5 504 DNA Haemophilus influenzae 5atgccattac ttgatagttt taaagtggat cacacaaaaa tgaacgcacc tgcagtacgc 60attgcaaaaa cgatgctcac gccaaaaggc gataatatta ctgtttttga tttacgtttt 120tgtattccaa acaaagaaat tctttcccca aaaggcattc atacacttga acatttattt 180gctggattta tgcgcgatca tttaaatggc gatagcatag aaattattga tatttctccg 240atgggatgtc gcacgggatt ttatatgtct ttgattggca caccaaatga acagaaagtg 300tctgaggctt ggttagcttc aatgcaagat gttttaggtg tacaagatca agcttctatt 360cctgaattaa atatctatca atgcggaagc tatacggaac attccttaga agatgcacac 420gaaattgcca aaaatgttat cgcacgcggt ataggtgtaa ataaaaatga agatttgtca 480ctcgataatt ccttattaaa atag 504 6 468 DNA Helicobacter pylori 6atgaaaacac caaaaatgaa tgtagagagt tttaatttgg atcacaccaa agtcaaagcc 60ccttatgtgc gtgtcgctga tcgcaaaaag ggcgttaatg gggatttgat tgtcaaatac 120gatgtgcgct tcaagcagcc caaccaagat cacatggaca tgcctagcct acattcttta 180gagcatttag tcgctgaaat tatccgcaac catgccagtt atgtcgtgga ttggtcgcct 240atgggttgcc aaacgggatt ttatctcaca gtgttaaacc atgacaatta cacagagatt 300ttagaggttt tagaaaagac catgcaagat gtgttaaagg ctacagaagt gcctgccagc 360aatgaaaagc aatgcggttg ggcggctaac cacactttag agggtgctaa ggatttagcg 420cgcgcttttt tagacaaacg cgctgagtgg tctgaagtgg gggtttga 468 7 482 DNABacillus subtilis 7 atgccttcag tagaaagttt tgagcttgat cataatgcggttgttgctcc atatgtaaga 60 cattgcggcg tgcataaagt gggaacagac ggcgttgtaaataaatttga cattcgtttt 120 tgccagccaa ataaacaggc gatgaagcct gacaccattcacacactcga gcatttgctc 180 gcgtttacga ttcgttctca cgctgagaaa tacgatcattttgatatcat tgatatttct 240 ccaatgggct gccagacagg ctattatcta gttgtgagcggagagccgac atcagcggaa 300 atcgttgatc tgcttgaaga cacaatgaag gaagcggtagagattacaga aatacctgct 360 gcgaatgaaa agcagtgcgg ccaagcgaag cttcatgatctggaaggcgc taaacgttta 420 atgcgtttct ggctttcaca ggataaagaa gaattgctaaaagtatttgg ctaaaataga 480 aa 482 8 537 DNA Borrelia burdorferi 8atgaatttga atgggaaaaa ttagattttg taaaaaaaat acaaacagcg ctaaaaaaat 60gaaaaaaata acaagcttta caatagatca tacaaaactc aaccctggca tatatgtctc 120aagaaaagat acctttgaaa atgtaatatt tactacaata gacattagaa tcaaagctcc 180caacatcgaa ccaataattg aaaacgcagc aatacataca atagagcaca taggagctac 240tttacttaga aataatgaag tttggaccga aaaaatagta tattttggcc ctatgggatg 300cagaactggt ttttacttaa taatttttgg agactatgaa agtaaagatc ttgttgactt 360agtctcatgg cttttttccg aaatcgtaaa tttttcagaa cctatcccag gcgcaagtga 420taaggaatgc ggaaattaca aagaacataa ccttgatatg gctaaatatg aatcttctaa 480atacttacaa atattaaaca atattaaaga agaaaattta aaatatcctt agctcat 537 9 519DNA Vibrio cholerae 9 atgccattat tagacagttt taccgtcgat catactcgtatgaatgcacc ggcggtgcgt 60 gttgccaaaa ccatgcaaac cccaaaaggg gatacgattaccgtatttga tttgcgtttt 120 actatgccaa acaaagatat cttgtctgag cgcggtatccatactctaga gcatctctac 180 gcgggcttta tgcgcaatca ccttaacggc agccaagtggagatcatcga tatttcacca 240 atgggttgcc gtacaggttt ctacatgagc ttgattggtgcgccgacaga acagcaagtg 300 gcacaagcat ggctagccgc aatgcaagat gtgttgaaagttgaaagcca agagcaaatt 360 cctgagctga atgagtacca gtgcggcact gcggcgatgcactcgctcga agaagccaaa 420 gcgattgcga aaaacgtgat tgcggcaggc atctcggttaaccgtaacga tgagttggcg 480 ctgcccgaat ctatgctcaa tgagctgaag gttcactaa 51910 172 PRT Vibrio harveyi 10 Met Pro Leu Leu Asp Ser Phe Thr Val Asp HisThr Arg Met Asn Ala 1 5 10 15 Pro Ala Val Arg Val Ala Lys Thr Met GlnThr Pro Lys Gly Asp Thr 20 25 30 Ile Thr Val Phe Asp Leu Arg Phe Thr AlaPro Asn Lys Asp Ile Leu 35 40 45 Ser Glu Lys Gly Ile His Thr Leu Glu HisLeu Tyr Ala Gly Phe Met 50 55 60 Arg Asn His Leu Asn Gly Asp Ser Val GluIle Ile Asp Ile Ser Pro 65 70 75 80 Met Gly Cys Arg Thr Gly Phe Tyr MetSer Leu Ile Gly Thr Pro Ser 85 90 95 Glu Gln Gln Val Ala Asp Ala Trp IleAla Ala Met Glu Asp Val Leu 100 105 110 Leu Val Glu Asn Gln Asn Lys IlePro Glu Leu Asn Glu Tyr Gln Cys 115 120 125 Gly Thr Ala Ala Met His SerLeu Asp Glu Ala Lys Gln Ile Ala Lys 130 135 140 Asn Ile Leu Glu Val GlyVal Ala Val Asn Lys Asn Asp Glu Leu Ala 145 150 155 160 Leu Pro Glu SerMet Leu Arg Glu Leu Arg Ile Asp 165 170 11 171 PRT Escherichia coli 11Met Pro Leu Leu Asp Ser Phe Thr Val Asp His Thr Arg Met Glu Ala 1 5 1015 Pro Ala Val Arg Val Ala Lys Thr Met Asn Thr Pro His Gly Asp Ala 20 2530 Ile Thr Val Phe Asp Leu Arg Phe Cys Val Pro Asn Lys Glu Val Met 35 4045 Pro Glu Arg Gly Ile His Thr Leu Glu His Leu Phe Ala Gly Phe Met 50 5560 Arg Asn His Leu Asn Gly Asn Gly Val Glu Ile Ile Asp Ile Ser Pro 65 7075 80 Met Gly Cys Ala Thr Gly Phe Tyr Met Ser Leu Ile Gly Thr Pro Asp 8590 95 Glu Gln Arg Val Ala Asp Ala Trp Lys Ala Ala Met Glu Asp Val Leu100 105 110 Lys Val Gln Asp Gln Asn Gln Ile Pro Glu Leu Asn Val Tyr GlnCys 115 120 125 Gly Thr Tyr Gln Met His Ser Leu Gln Glu Ala Gln Asp IleAla Arg 130 135 140 Ser Ile Leu Glu Arg Asp Val Arg Ile Asn Ser Asn GluGlu Leu Ala 145 150 155 160 Leu Pro Lys Glu Lys Leu Gln Glu Leu His Ile165 170 12 164 PRT Salmonella typhimurium VARIANT (0)...(0) starts withresidue 8 and has 3 frameshifts 12 Asn Ser Asp His Thr Arg Met Gln AlaPro Ala Val Arg Val Ala Lys 1 5 10 15 Thr Met Asn Thr Pro His Gly AspAla Ile Thr Val Phe Asp Leu Arg 20 25 30 Phe Cys Ile Pro Asn Lys Glu ValMet Pro Glu Lys Gly Ile His Thr 35 40 45 Leu Glu His Leu Phe Ala Gly PheMet Arg Asp His Leu Asn Gly Asn 50 55 60 Gly Val Glu Ile Ile Asp Ile SerPro Met Gly Cys Arg Thr Gly Phe 65 70 75 80 Tyr Met Ser Leu Ile Gly ThrPro Asp Glu Gln Arg Val Ala Asp Ala 85 90 95 Trp Leu Ala Ala Met Ala AspVal Leu Lys Val Gln Asp Gln Asn Gln 100 105 110 Ile Pro Glu Leu Asn ValTyr Gln Cys Gly Thr Tyr Gln Met His Ser 115 120 125 Leu Ser Glu Ala GlnAsp Ile Ala Arg His Ile Leu Glu Arg Asp Val 130 135 140 Arg Val Asn SerAsn Lys Glu Leu Ala Leu Pro Lys Glu Lys Leu Gln 145 150 155 160 Glu ThrAsp Ile 13 167 PRT Haemophilus influenzae 13 Met Pro Leu Leu Asp Ser PheLys Val Asp His Thr Lys Met Asn Ala 1 5 10 15 Pro Ala Val Arg Ile AlaLys Thr Met Leu Thr Pro Lys Gly Asp Asn 20 25 30 Ile Thr Val Phe Asp LeuArg Phe Cys Ile Pro Asn Lys Glu Ile Leu 35 40 45 Ser Pro Lys Gly Ile HisThr Leu Glu His Leu Phe Ala Gly Phe Met 50 55 60 Arg Asp His Leu Asn GlyAsp Ser Ile Glu Ile Ile Asp Ile Ser Pro 65 70 75 80 Met Gly Cys Arg ThrGly Phe Tyr Met Ser Leu Ile Gly Thr Pro Asn 85 90 95 Glu Gln Lys Val SerGlu Ala Trp Leu Ala Ser Met Gln Asp Val Leu 100 105 110 Gly Val Gln AspGln Ala Ser Ile Pro Glu Leu Asn Ile Tyr Gln Cys 115 120 125 Gly Ser TyrThr Glu His Ser Leu Glu Asp Ala His Glu Ile Ala Lys 130 135 140 Asn ValIle Ala Arg Gly Ile Gly Val Asn Lys Asn Glu Asp Leu Ser 145 150 155 160Leu Asp Asn Ser Leu Leu Lys 165 14 155 PRT Helicobacter pylori 14 MetLys Thr Pro Lys Met Asn Val Glu Ser Phe Asn Leu Asp His Thr 1 5 10 15Lys Val Lys Ala Pro Tyr Val Arg Val Ala Asp Arg Lys Lys Gly Val 20 25 30Asn Gly Asp Leu Ile Val Lys Tyr Asp Val Arg Phe Lys Gln Pro Asn 35 40 45Gln Asp His Met Asp Met Pro Ser Leu His Ser Leu Glu His Leu Val 50 55 60Ala Glu Ile Ile Arg Asn His Ala Ser Tyr Val Val Asp Trp Ser Pro 65 70 7580 Met Gly Cys Gln Thr Gly Phe Tyr Leu Thr Val Leu Asn His Asp Asn 85 9095 Tyr Thr Glu Ile Leu Glu Val Leu Glu Lys Thr Met Gln Asp Val Leu 100105 110 Lys Ala Thr Glu Val Pro Ala Ser Asn Glu Lys Gln Cys Gly Trp Ala115 120 125 Ala Asn His Thr Leu Glu Gly Ala Lys Asp Leu Ala Arg Ala PheLeu 130 135 140 Asp Ile Arg Ala Glu Trp Ser Glu Val Gly Val 145 150 15515 157 PRT Bacillus subtilis 15 Met Pro Ser Val Glu Ser Phe Glu Leu AspHis Asn Ala Val Val Ala 1 5 10 15 Pro Tyr Val Arg His Cys Gly Val HisLys Val Gly Thr Asp Gly Val 20 25 30 Val Asn Lys Phe Asp Ile Arg Phe CysGln Pro Asn Lys Gln Ala Met 35 40 45 Lys Pro Asp Thr Ile His Thr Leu GluHis Leu Leu Ala Phe Thr Ile 50 55 60 Arg Ser His Ala Glu Lys Tyr Asp HisPhe Asp Ile Ile Asp Ile Ser 65 70 75 80 Pro Met Gly Cys Gln Thr Gly TyrTyr Leu Val Val Ser Gly Glu Pro 85 90 95 Thr Ser Ala Glu Ile Val Asp LeuLeu Glu Asp Thr Met Lys Glu Ala 100 105 110 Val Glu Ile Thr Glu Ile ProAla Ala Asn Glu Lys Gln Cys Gly Gln 115 120 125 Ala Lys Leu His Asp LeuGlu Gly Ala Lys Arg Leu Met Arg Phe Trp 130 135 140 Leu Ser Gln Asp LysGlu Glu Leu Leu Lys Val Phe Gly 145 150 155 16 173 PRT Borreliaburgdorferi 16 Met Gly Lys Ile Arg Phe Cys Lys Lys Asn Thr Asn Ser AlaLys Lys 1 5 10 15 Met Lys Leu Ile Thr Ser Phe Thr Ile Asp His Thr LysLeu Asn Pro 20 25 30 Gly Ile Tyr Val Ser Arg Lys Asp Thr Phe Glu Asn ValIle Phe Thr 35 40 45 Thr Ile Asp Ile Arg Ile Lys Ala Pro Asn Ile Glu ProIle Ile Glu 50 55 60 Asn Ala Ala Ile His Thr Ile Glu His Ile Gly Ala ThrLeu Leu Arg 65 70 75 80 Asn Asn Glu Val Trp Thr Glu Lys Ile Val Tyr PheGly Pro Met Gly 85 90 95 Cys Arg Thr Gly Phe Tyr Leu Ile Ile Phe Gly AspTyr Glu Ser Lys 100 105 110 Asp Leu Val Asp Leu Val Ser Trp Leu Phe SerGlu Ile Val Asn Phe 115 120 125 Ser Glu Pro Ile Pro Gly Ala Ser Asp LysGlu Cys Gly Asn Tyr Lys 130 135 140 Glu His Asn Leu Asp Met Ala Lys TyrGlu Ser Ser Lys Leu Tyr Gln 145 150 155 160 Ile Leu Asn Asn Ile Lys GluGlu Asn Leu Lys Tyr Pro 165 170 17 172 PRT Vibrio cholerae 17 Met ProLeu Leu Asp Ser Phe Thr Val Asp His Thr Arg Met Asn Ala 1 5 10 15 ProAla Val Arg Val Ala Lys Thr Met Gln Thr Pro Lys Gly Asp Thr 20 25 30 IleThr Val Phe Asp Leu Arg Phe Thr Met Pro Asn Lys Asp Ile Leu 35 40 45 SerGlu Arg Gly Ile His Thr Leu Glu His Leu Tyr Ala Gly Phe Met 50 55 60 ArgAsn His Leu Asn Gly Ser Gln Val Glu Ile Ile Asp Ile Ser Pro 65 70 75 80Met Gly Cys Arg Thr Gly Phe Tyr Met Ser Leu Ile Gly Ala Pro Thr 85 90 95Glu Gln Gln Val Ala Gln Ala Trp Leu Ala Ala Met Gln Asp Val Leu 100 105110 Lys Val Glu Ser Gln Glu Gln Ile Pro Glu Leu Asn Glu Tyr Gln Cys 115120 125 Gly Thr Ala Ala Met His Ser Leu Glu Glu Ala Lys Ala Ile Ala Lys130 135 140 Asn Val Ile Ala Ala Gly Ile Ser Val Asn Arg Asn Asp Glu LeuAla 145 150 155 160 Leu Pro Glu Ser Met Leu Asn Glu Leu Lys Val His 165170

What is claimed is:
 1. An isolated bacterial extracellular signalingfactor comprising at least one molecule that is polar and uncharged, andhaving an approximate molecular weight of less than 1,000 kDa, whereinthe factor interacts with LuxQ protein thereby inducing expression of aVibrio harveyi operon comprising luminescence genes luxCDABE.
 2. Thefactor of claim 1, having a specific activity wherein about 0.1 to 1.0mg of a preparation of the factor stimulates about a 1,000-fold increasein luminescence, as measured in a bioassay using a V. harveyi Sensor 2+reporter strain.
 3. The factor of claim 1, having a specific activitywherein about 1 to 10 μg of a preparation of the factor stimulates abouta 1,000-fold increase in luminescence, as measured in a bioassay using aV. harveyi Sensor 2+ reporter strain.
 4. The factor of claim 1, producedby a bacterial cell selected from the group consisting of Vibrioharveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus,Pseudomonas phosphoreum, Yersinia enterocolitica, Escherichia coli,Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori,Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis,Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni,Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcusfaecalis, Streptococcus pneumoniae, Streptococcus pyogenes andStaphylococcus aureus.
 5. An isolated bacterial signaling factorcomprising a factor of the formula:


6. The factor of claim 5, wherein the factor is produced by a bacterialcell selected from the group consisting of Vibrio harveyi, Vibriocholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonasphosphoreum, Yersinia enterocolitica, Escherichia coli, Salmonellatyphimurium, Haemophilus influenzae, Helicobacter pylori, Bacillussubtilis, Borrelia burgfdorferi, Neisseria meningitidis, Neisseriagonorrhoeae, Yersinia pestis, Campylobacter jejuni, Deinococcusradiodurans, Mycobacterium tuberculosis, Enterococcus faecalis,Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcusaureus.
 7. An optically active isomer of a factor having the formula asdefined in claim
 5. 8. The optically active isomer of claim 7, whereinthe isomer is the L-isomer.
 9. The optically active isomer of claim 7,wherein the isomer is the D-isomer.
 10. An isolated bacterial signalingfactor comprising a factor having the formula:

wherein R1, R2, R3 and R4 are independently selected from the groupconsisting of hydrido, halo, alkyl, haloalkyl, cycloalkyl, cycloalkenyl,heterocyclyl, methyl, cyano, alkoxycarbonyl, amino, carboxyl, hydroxyl,formyl, nitro, fluoro, chloro, bromo, methyl, aryl, heteroaryl, aralkyl,heteroarylalkyl, alkylsulfonyl, haloalkylsulfonyl, arylsulfonyl,heteroarylsulfonyl, hydroxyalkyl, mercaptoalkyl, alkoxyalkyl,aryloxyalkyl, heteroaryloxyalkyl, aralkyloxyalkyl,heteroarylalkyloxyalkyl, alkylthioalkyl, arylthioalkyl,heteroarylthioalkyl, aralkylthioalkyl, heteroarylalkylthioalkyl,haloalkylcarbonyl, haloalkyl(hydroxy)alkyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl,carboxyalkyl, alkoxycarbonylalkyl, alkylcarbonyloxyalkyl, aminoalkyl,alkylaminoalkyl, arylaminoalkyl, aralkylaminoalkyl,heteroarylaminoalkyl, heteroarylalkylaminoalkyl, alkoxy, and aryloxy;phenyl, cyclohexyl, cyclohexenyl, benzofuryl, benzodioxolyl, furyl,imidazolyl, thienyl, thiazolyl, pyrrolyl, oxazolyl, isoxazolyl,triazolyl, pyrimidinyl, isoquinolyl, quinolinyl, benzimidazolyl,indolyl, pyrazolyl and pyridyl, aminosulfonyl, fluoro, chloro, bromo,methylthio, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl,hexyl, cyano, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl,tert-butoxycarbonyl, propoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,pentoxycarbonyl, methylcarbonyl, fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl,dichloropropyl, methoxy, methylenedioxy, ethoxy, propoxy, n-butoxy,hydroxymethyl, hydroxyethyl, methoxymethyl, ethoxymethyl,trifluoromethoxy, methylamino, N,N-dimethylamino, phenylamino,ethoxycarbonylethyl, and methoxycarbonylmethyl, methyl, ethyl,fluoromethyl, difluoromethyl, trifluoromethyl, cyano, methoxycarbonyl,ethoxycarbonyl, tert-butoxycarbonyl, benzyl, phenylethyl, phenylpropyl,methylsulfonyl, phenylsulfonyl, trifluoromethylsulfonyl, hydroxymethyl,hydroxyethyl, methoxymethyl, ethoxymethyl, methylcarbonyl,ethylcarbonyl, trifluoromethylcarbonyl, trifluoro(hydroxy)ethyl,phenylcarbonyl, benzylcarbonyl, methoxycarbonylmethyl,ethoxycarbonylethyl, carboxymethyl, carboxypropyl,methylcarbonyloxymethyl, phenyloxy, phenyloxymethyl, thienyl, furyl, andpyridyl, wherein the thienyl, furyl, pyridyl, methylthio,methylsulfinyl, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl,hexyl, cyano, fluoromethyl, difluoromethyl, trifluoromethyl,chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl,heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl,difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, methoxy,methylenedioxy, ethoxy, propoxy, n-butoxy, hydroxymethyl, hydroxyethyland trifluoromethoxy.
 11. An optically active isomer of a factor havingthe formula as defined in claim
 10. 12. The optically active isomer ofclaim 11, wherein the isomer is the L-isomer.
 13. The optically activeisomer of claim 11, wherein the isomer is the D-isomer.
 14. A method foridentifying a compound that regulates the activity of a signaling factorcomprising: a) contacting the signaling factor with the compound; b)measuring the activity of the signaling factor in the presence of thecompound and comparing the activity of the signaling factor obtained inthe presence of the compound to the activity of the signaling factorobtained in the absence of the compound; and c) identifying a compoundthat regulates the activity of the signaling factor.
 15. The method ofclaim 14, wherein the signaling factor is autoinducer-2.
 16. The methodof claim 15, wherein the autoinducer-2 is4,5-dihydroxy-2,3-pentanedione.
 17. The method of claim 15, wherein theautoinducer-2 is homocysteine.
 18. The method of claim 14, wherein thecontacting is in vivo.
 19. The method of claim 14, wherein thecontacting is in vitro.
 20. The method of claim 14, wherein theregulation is by increasing the activity of the signaling factor. 21.The method of claim 14, wherein the regulation is by decreasing theactivity of the signaling factor.
 22. The method of claim 14, whereinthe compound is a polypeptide.
 23. The method of claim 14, wherein thecompound is a small molecule.
 24. A method for detecting an autoinducermolecule in a sample comprising: a) contacting the sample with abacterial cell, or extract thereof, comprising biosynthetic pathwaysthat produce a detectable amount of light in response to an exogenousautoinducer, the bacterial cell having at least two distinct alterationsin gene loci that participate in autoinducer pathways, wherein a firstalteration in a gene locus comprises an alteration that inhibitsdetection of a first autoinducer and wherein a second alteration in agene locus comprises an alteration that inhibits production of a secondautoinducer; and b) measuring light produced by the bacterial cell, orextract thereof, of a).
 25. The method of claim 24, wherein the firstalteration in a gene locus comprises an alteration in the LuxN gene. 26.The method of claim 24, wherein the first alteration in a gene locusinhibits detection of autoinducer-1.
 27. The method of claim 24, whereinthe second alteration in a gene locus comprises an alteration in theLuxS gene.
 28. The method of claim 24, wherein the second alteration ina gene locus inhibits production of endogenous autoinducer-2.
 29. Themethod of claim 24, wherein the amount of light measured in the presenceof the sample is greater than the amount of light measured in theabsence of the sample is indicative of the presence of an autoinducer inthe sample.
 30. The method of claim 24, wherein the sample is selectedfrom a biological fluid, tissue homogenate or medium conditioned by thegrowth of a test bacterial cell suspected of autoinducer production. 31.The method of claim 24, wherein the exogenous autoinducer is produced bya bacterial cell selected from the group consisting of Vibrio harveyi,Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus,Pseudomonas phosphoreum, Yersinia enterocolitica, Escherichia coli,Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori,Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis,Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni,Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcusfaecalis, Streptococcus pneumoniae, Streptococcus pyogenes andStaphylococcus aureus.
 32. The method of claim 24, wherein the exogenousautoinducer is autoinducer-2.
 33. The method of claim 24, wherein thebacterial cell is a mutant of a parent strain that is bioluminescent andcapable of producing an autoinducer.
 34. The method of claim 33, whereinthe parent strain is V. harveyi.
 35. The method of claim 34, wherein thebacterial cell is V. harveyi strain MM32.
 36. A bacterial cellcomprising at least two distinct alterations in gene loci thatparticipate in autoinducer pathways, wherein a first alteration in agene locus comprises an alteration that inhibits detection of a firstautoinducer and wherein a second alteration in a gene locus comprises analteration that inhibits production of a second autoinducer and whereinthe cell is bioluminescent when contacted with an autoinducer.
 37. Thecell of claim 36, wherein the first alteration in a gene locus comprisesan alteration in the LuxN gene.
 38. The cell of claim 36, wherein thefirst alteration in a gene locus inhibits detection of autoinducer-1.39. The cell of claim 36, wherein the second alteration in a gene locuscomprises an alteration in the LuxS gene.
 40. The cell of claim 36,wherein the second alteration in a gene locus inhibits production ofendogenous autoinducer-2.
 41. The method of claim 24, wherein thebacterial cell is a mutant of a V. harveyi that is bioluminescent andcapable of producing an autoinducer.
 42. The cell of claim 41, whereinthe bacterial cell is V. harveyi strain MM32.
 43. A method foridentifying an autoinducer analog that regulates the activity of anautoinducer, comprising: a) contacting a bacterial cell, or extractthereof, comprising biosynthetic pathways which will produce adetectable amount of light in response to an autoinducer with anautoinducer analog; b) comparing the amount of light produced by thebacterial cell, or extract thereof, in the presence of an autoinducerwith the amount produced in the presence of the autoinducer analog,wherein a change in the production of light is indicative of anautoinducer analog that regulates the activity of an autoinducer. 44.The method of claim 43, wherein the autoinducer is endogenousautoinducer.
 45. The method of claim 43, wherein the autoinducer isexogenous autoinducer.
 46. The method of claim 43, wherein theautoinducer is autoinducer-2.
 47. The method of claim 43, wherein thecontacting is in vitro.
 48. The method of claim 43, wherein thecontacting is in vivo.
 49. The method of claim 43, wherein theregulation is by inhibition of autoinducer activity.
 50. The method ofclaim 43, wherein the regulation is by enhancement of autoinduceractivity.
 51. The method of claim 43, wherein the analog comprises aribose derivative.
 52. The method of claim 43, wherein the bacterialcell further comprises at least one distinct alteration in a gene locusthat participates in an autoinducer pathway, wherein the alterationinhibits the production or detection of an autoinducer.
 53. The methodof claim 52, wherein the alteration in a gene locus comprises analteration in the LuxS gene.
 54. The method of claim 52, wherein thealteration in a gene locus inhibits production of endogenousautoinducer-2.
 55. The method of claim 52, wherein the alteration in agene locus comprises an alteration in the LuxN gene.
 56. The method ofclaim 52, wherein the alteration in a gene locus inhibits detection ofautoinducer-1.
 57. The method of claim 49, wherein the alteration is inthe LuxN and LuxS loci.
 58. The method of claim 49, wherein thebacterial cell is V. harveyi strain MM32.
 59. A method for producingautoinducer-2 comprising contacting S-adenosylhomocysteine (SAH) with aLuxS protein under conditions and for such time as to promote theconversion of S-adenosylhomocysteine to autoinducer-2.
 60. The method ofclaim 59, wherein the production of autoinducer-2 is in vitro.
 61. Themethod of claim 59, wherein the production of autoinducer-2 is in vivo.62. The method of claim 59, further comprising a5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase protein(pfs).
 63. The method of claim 62, wherein the autoinducer-2 is4,5-dihydroxy-2,3-pentanedione.
 64. A method for producing autoinducer-2comprising contacting S-ribosylhomocysteine (SRH) with a LuxSpolypeptide under conditions and for such time as to promote theconversion of S-ribosylhomocysteine to autoinducer-2.
 65. The method ofclaim 64, wherein the autoinducer-2 is 4,5-dihydroxy-2,3-pentanedione.66. A method for producing autoinducer-2 comprising: a) contactingS-adenosylhomocysteine (SAH) with a5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (pfs) proteinunder conditions and for such time as to promote the conversion ofS-adenosylhomocysteine to S-ribosylhomocysteine; b) contacting theS-ribosylhomocysteine from a) with a LuxS protein under conditions andfor such time as to promote the conversion of S-ribosylhomocysteine toautoinducer-2.
 67. The method of claim 66, wherein the autoinducer-2 is4,5-dihydroxy-2,3-pentanedione.
 68. A method for detecting anautoinducer-associated bacterial biomarker comprising; a) contacting atleast one bacterial cell with an autoinducer molecule under conditionsand for such time as to promote induction of a bacterial biomarker; andb) detecting the bacterial biomarker.
 69. The method of claim 68,wherein the autoinducer is autoinducer-2.
 70. The method of claim 69,wherein the autoinducer-2 is 4,5-dihydroxy-2,3-pentanedione.
 71. Themethod of claim 68, wherein the biomarker is a nucleic acid.
 72. Themethod of claim 68, wherein the biomarker is a protein.
 73. The methodof claim 68, wherein the biomarker is an antigen.
 74. The method ofclaim 73, wherein the antigen is indicative of bacterial pathogenicity.75. The method of claim 68, wherein the biomarker is a phosphorylatedprotein.
 76. The method of claim 68, wherein the detection is by aprobe.
 77. The method of claim 76, wherein the probe is a nucleic acid.78. The method of claim 76, wherein the probe is an antibody.
 79. Themethod of claim 78, wherein the antibody is polyclonal.
 80. The methodof claim 78, wherein the antibody is monoclonal.
 81. The method of claim76, wherein the probe is detectably labeled.
 82. The method of claim 68,wherein the bacterial cell is selected from the group consisting ofVibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrioalginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica,Escherichia coli, Salmonella typhimurium, Haemophilus influenzae,Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi, Neisseriameningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacterjejuni, Deinococcus radiodurans, Mycobacterium tuberculosis,Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenesand Staphylococcus aureus.
 83. A method for detecting a target compoundthat binds to a LuxP protein comprising contacting the LuxP protein withthe target compound and detecting binding of the compound to LuxP. 84.The method of claim 83, wherein the target compound is autoinducer-2.85. The method of claim 83, wherein the target compound is anautoinducer-2 analog.
 86. The method of claim 83, wherein the detectingis in vivo.
 87. The method of claim 83, wherein the detecting is invitro.
 88. A method for regulating bacterial biofilm formationcomprising contacting a bacterium capable of biofilm formation with acompound capable of regulating biofilm formation wherein the compoundregulates autoinducer-2 activity.
 89. The method of claim 88, whereinthe compound is an autoinducer-2 analog.
 90. The method of claim 88,wherein the compound is a polypeptide.
 91. The method of claim 88,wherein the compound is a small molecule.
 92. The method of claim 88,wherein the contacting is in vivo.
 93. The method of claim 88, whereinthe contacting is in vitro.
 94. The method of claim 88, wherein theregulating is by inhibiting biofilm formation.
 95. An isolated nucleicacid molecule that encodes a protein necessary for biosynthesis of abacterial extracellular signaling factor, wherein said factor interactswith LuxQ protein thereby inducing expression of a Vibrio harveyi operoncomprising luminescence genes luxCDABE.
 96. The nucleic acid molecule ofclaim 95, isolated from a bacterial cell selected from the groupconsisting of Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio alginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica,Escherichia coli, Salmonella typhimurium, Haemophilus influenzae,Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi, Neisseriameningitidis; Neisseria gonorrhoeae, Yersinia pestis, Campylobacterjejuni, Deinococcus radiodurans, Mycobacterium tuberculosis,Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenesand Staphylococcus aureus.
 97. The nucleic acid molecule of claim 95,encoding a polypeptide having between about 150 and 200 amino acidresidues.
 98. The nucleic acid molecule of claim 95, wherein the encodedpolypeptide comprises an amino acid sequence substantially the same as asequence selected from the group consisting of SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16 SEQ ID NO:17 and a consensus sequence derived from a comparison oftwo or more of SEQ ID NOS: 10-17.
 99. The nucleic acid molecule of claim95, having a sequence substantially the same as a sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9and a consensus sequence derived from a comparison of two or more of SEQID NOS: 1-9.
 100. A recombinant DNA molecule, comprising a vector havingan insert that includes the nucleic acid molecule of claim
 95. 101. Apolypeptide produced by expression of the nucleic acid molecule of claim95.
 102. An isolated nucleic acid molecule having a sequence selectedfrom the group consisting of: a) SEQ ID NO:1; b) a variant of SEQ IDNO:1; c) a natural mutant of SEQ ID NO:1; d) a sequence hybridizing withSEQ ID NO:1 or its complement and encoding a polypeptide substantiallythe same as the polypeptide encoded by SEQ ID NO:1; and e) a sequenceencoding part or all of a polypeptide having amino acid SEQ ID NO:10.103. An isolated nucleic acid molecule having a sequence selected fromthe group consisting of: a) SEQ ID NO:2; b) a variant of SEQ ID NO:2; c)a natural mutant of SEQ ID NO:2; d) a sequence hybridizing with SEQ IDNO:2 or its complement and encoding a polypeptide substantially the sameas the polypeptide encoded by SEQ ID NO:2; and e) a sequence encodingpart or all of a polypeptide having amino acid SEQ ID NO:11.
 104. Anisolated nucleic acid molecule having a sequence selected from the groupconsisting of: a) SEQ ID NO:4; b) a variant of SEQ ID NO:4; c) a naturalmutant of SEQ ID NO:4; d) a sequence hybridizing with SEQ ID NO:4 or itscomplement and encoding a polypeptide substantially the same as thepolypeptide encoded by SEQ ID NO:4; and e) a sequence encoding part orall of a polypeptide having amino acid SEQ ID NO:12.
 105. A recombinantDNA molecule comprising a vector and an insert that includes the nucleicacid molecule of claim 102, 103 or
 104. 106. A polypeptide produced byexpression of the nucleic acid molecule of claim 102, 103 or
 104. 107. Amethod of purifying a bacterial extracellular signaling factor whereinsaid factor interacts with LuxQ protein thereby inducing expression of aVibrio harveyi operon comprising luminescence genes luxCDABE,comprising: a) growing, in a culture medium, bacterial cells thatproduce the signaling molecule; b) separating the bacterial cells fromthe culture medium; c) incubating the bacterial cells in a solutionhaving high osmolarity, under conditions that permit production andsecretion of the signaling molecule from the bacterial cells; d)separating the bacterial cells from the high osmolarity solution; and e)purifying the factor from the high osmolarity solution.
 108. The methodof claim 107, which further comprises a) separating polar compounds fromnon-polar compounds in an evaporated sample of the high osmolaritysolution; and b) subjecting the polar compounds to reverse-phase HighPerformance Liquid Chromatography.
 109. The method of claim 107, whereinthe high osmolarity solution comprises at least 0.4 M monovalent salt.110. The method of claim 109, comprising 0.4-0.5 M NaCl.
 111. The methodof claim 107, which further comprises growing the bacterial cells in aculture medium containing a carbohydrate selected from the groupconsisting of glucose, fructose, mannose, glucitol, glucosamine,galactose and arabinose.
 112. A purified signaling molecule produced bythe method of claim
 107. 113. A kit comprising a carrier means beingcompartmentalized to receive in close confinement one or more containermeans containing a strain of bacteria, or extract thereof, comprisingbiosynthetic pathways that produce a detectable amount of light inresponse to an exogenous autoinducer, the bacterial cell having at leasttwo distinct alterations in gene loci that participate in autoinducerpathways, wherein a first alteration in a gene locus comprises analteration that inhibits detection of a first autoinducer and wherein asecond alteration in a gene locus comprises an alteration that inhibitsproduction of a second autoinducer.
 114. The kit of claim 113, whereinthe first alteration in a gene locus comprises an alteration in the LuxNgene.
 115. The method of claim 113, wherein the first alteration in agene locus inhibits detection of autoinducer-1.
 116. The method of claim113, wherein the second alteration in a gene locus comprises analteration in the LuxS gene.
 117. The method of claim 113, wherein thesecond alteration in a gene locus inhibits production of endogenousautoinducer-2.